CN105330705A - Method for preparation of tetrasaccharide lacto-N-neotetraose (LNNT) containing N-acetyllactosamine - Google Patents

Method for preparation of tetrasaccharide lacto-N-neotetraose (LNNT) containing N-acetyllactosamine Download PDF

Info

Publication number
CN105330705A
CN105330705A CN201510751641.5A CN201510751641A CN105330705A CN 105330705 A CN105330705 A CN 105330705A CN 201510751641 A CN201510751641 A CN 201510751641A CN 105330705 A CN105330705 A CN 105330705A
Authority
CN
China
Prior art keywords
general formula
lnnt
compound
3galp
4glcnacp
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201510751641.5A
Other languages
Chinese (zh)
Inventor
伊什特万·鲍伊佐
久洛·戴卡尼
卡罗伊·阿戈什顿
伊格纳西奥·费格罗拉·佩雷斯
朱莉安·布泰
马库斯·赫德罗斯
费伦茨·霍瓦特
皮罗什卡·科瓦奇-彭泽什
拉斯·克罗杰
克里斯托夫·勒里希
安德烈亚斯·施罗温
扬尼斯·弗拉斯达斯
彼得·特林卡
拉斯洛·卡尔马尔
伊姆雷·科瓦奇
尚多尔·德姆科
阿格尼斯·阿戈什顿
克里斯蒂安·里辛格
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Glycom AS
Original Assignee
Glycom AS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Glycom AS filed Critical Glycom AS
Publication of CN105330705A publication Critical patent/CN105330705A/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/30—Foods or foodstuffs containing additives; Preparation or treatment thereof containing carbohydrate syrups; containing sugars; containing sugar alcohols, e.g. xylitol; containing starch hydrolysates, e.g. dextrin
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
    • C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
    • C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L7/00—Cereal-derived products; Malt products; Preparation or treatment thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Abstract

The present invention relates to a method for preparation of the tetrasaccharide lacto-N-neotetraose (LNnt, formula (I)) especially in large scale, as well as intermediates in the synthesis, a new crystal form (polymorph) of LNnt, and the use thereof in pharmaceutical or nutritional compositions.

Description

For the preparation of the method for the tetrose lacto-N-neotetraose (LNNT) containing N-acetyl lactosamine
The application is international application no PCT/DK2011/050053, international filing date on February 21st, 2011, China's application number 201180009778.3, denomination of invention is the divisional application of the patent application of " method for the preparation of the tetrose lacto-N-neotetraose (LNNT) containing N-acetyl lactosamine ".
Technical field
The present invention relates to for the preparation of, prepare the method for Galp β 1-4GlcNAcp β 1-3Galp β 1-4Glc (LNnT) especially on a large scale, and the intermediate of LNnT and new crystalline form (polymorphic form).
Background technology
In the past between many decades, the preparation of human milk oligosaccharides (human milk oligosaccharides, HMO) and business-like interest are continued to increase.The importance of HMO directly to the biologic activity of its uniqueness as antibacterial, antiviral, immunity system is relevant with cognitive development enhanced activity.
Tetrose Galp β 1-4GlcNAcp β 1-3Galp β 1-4Glc (lacto-N-neotetraose, LNnT, scheme 1) is one of the oligosaccharides be present in human milk (Kuhn etc., Chem.Ber.1962,95,513 and 518, KobataMethodsEnzymol.1972,28,262].LNnT serves as the bacterial receptor of pneumococcus (pneumococci) and finds that it can be used for identifying the structure of the receptor-specific of glycosyltransferase, the substrate specificity of Glycosylase and antigenic determinant.In addition, LNnT represents the core texture element of the more complex oligosaccharide in the glycolipid and glycoprotein (intending globoside (paragloboside), 6-saliva base-LNnT etc.) with various physiologically active.
So far, also a large amount of LNnT can not be obtained by using separation, biotechnology and synthetic method.Owing to there is a large amount of similar oligosaccharides, even if so with milligram quantities be separated from human milk LNnT also quite difficulty.
The enzymic synthesis of LNnT is included under galactosyltransferase exists and is hatched by lactose-N-triose II UDP-semi-lactosi [J.Am.Chem.Soc.1986 such as Sabesan, 108,2068; EP-A-870841] or [Glycoconj.J.1999 such as Murata, 16,189 are hatched with lactose under beta-D-galactosidase exists; JP10-234394A].The enzymatic galactosylation of the suitable trisaccharide be combined with solid carrier is also described in detail [Carbohydr.Res.1999 such as Blixt, 319,80; The ibid.2004 such as Renaudie, 339,693].The enzymatic system code of these complexity is used for the very expensive method of the scale operation of LNnT and the purification schemes of difficulty.
The disclosed total synthesis program for LNnT [SovietJ.Bioorg.Chem.1978 such as Zurabyan, 4,679; The Carbohydr.Chem.1987 such as Paulsen, 169,105; The ibid.1999 such as Aly, 316,121] comprise multiple reactions steps, protecting group operation and chromatography purification, low-yield, and the LNnT of milligram quantities is only provided, therefore they do not provide attractive technology for extensive preparation.As intend globoside benzyl analogs 1-O-phenyl-LNnT synthesis also open TetrahedronLett.1978 such as [, 20,1717] Ponpipom.
When being separated Chem.Ber.1962 such as [, 95,513] Kuhn from natural origin or being prepared by enzyme method [EP-A-1405856], LNnT is characterized as being crystalline substance.
For LNnT with for its intermediate, still need to simplify expect up to now separation, purifying and preparation problem crystal product.
Summary of the invention
A first aspect of the present invention relates to a kind of method for the preparation of Galp β 1-4GlcNAcp β 1-3Galp β 1-4Glc (LNnT), said method comprising the steps of:
A) donor characterized by general formula 5 is made
Wherein R 4the optional acyl group replaced,
-NR 5r 6be selected from-NAc 2,-NH-halogenacyl, phthalimide, tetrachloro phthalimide, 2,3-diphenylmaleimide and 2,3-dimethylmaleimide,
X is selected from halogen ,-OC (=NH) CCl 3,-OAc ,-OBz and-SR 7, wherein R 7be selected from alkyl and the optional phenyl replaced,
React with the acceptor of general formula 6
Wherein R 1by the removable group of catalytic hydrogenolysis,
R 2the optional acyl group replaced, and
R 3be selected from acyl group or the H of optional replacement,
Thus produce the compound of general formula 4
Wherein R 1, R 2, R 3, R 4with-NR 5r 6as defined above,
B) by the converting compounds of general formula 4 be the compound of general formula 1
Wherein R 1as defined above,
C) compound crystal of described general formula 1 is made, and
D) make the compound of described general formula 1 through catalytic reduction subsequently.
A second aspect of the present invention provides a kind of polymorphic form of Galp β 1-4GlcNAcp β 1-3Galp β 1-4Glc (LNnT).
A third aspect of the present invention relates to the polymorphic form of Galp β 1-4GlcNAcp β 1-3Galp β 1-4Glc (LNnT), and it is used as pharmaceutical agent.
A fourth aspect of the present invention provides the polymorphic form of Galp β 1-4GlcNAcp β 1-3Galp β 1-4Glc (LNnT), and it is used as nutritional additive.
A fifth aspect of the present invention relates to a kind of pharmaceutical composition, and described pharmaceutical composition comprises the polymorphic form of Galp β 1-4GlcNAcp β 1-3Galp β 1-4Glc.
A sixth aspect of the present invention provides a kind of nutritive compositions, and described nutritive compositions comprises the polymorphic form of Galp β 1-4GlcNAcp β 1-3Galp β 1-4Glc.
A seventh aspect of the present invention relates to the compound of general formula 1 '
Wherein R ' 1be selected from substituted benzyl, the optional diphenyl-methyl replaced, the optional trityl replaced and the optional menaphthyl replaced.
A eighth aspect of the present invention provides the compound of general formula 2 '
Wherein R ' 1be selected from substituted benzyl, the optional diphenyl-methyl replaced, the optional trityl replaced and the optional menaphthyl replaced.
A ninth aspect of the present invention relates to the compound of general formula 3
Wherein R 1be selected from the optional benzyl replaced, the optional diphenyl-methyl replaced, the trityl optionally replaced and the optional menaphthyl replaced, and-NR 5r 6be selected from-NH-halogenacyl, phthalimide, tetrachloro phthalimide, 2,3-diphenylmaleimide and 2,3-dimethylmaleimide.
A tenth aspect of the present invention provides the compound of general formula 4 '
Wherein R 1be selected from the optional benzyl replaced, the optional diphenyl-methyl replaced, the trityl optionally replaced and the optional menaphthyl replaced, and R 2and R 4the optional acyl group replaced independently of one another, R 3be selected from acyl group and the H ,-NR of optional replacement 5r 6be selected from-NAc 2,-NH-halogenacyl, tetrachloro phthalimide, 2,3-diphenylmaleimide and 2,3-dimethylmaleimide.
A eleventh aspect of the present invention relates to the compound of general formula 4a
Wherein R 1be selected from the optional benzyl replaced, the optional diphenyl-methyl replaced, the trityl optionally replaced and the optional menaphthyl replaced.
A twelveth aspect of the present invention provides the compound of general formula 5 '
Wherein R 4the optional acyl group replaced, preferred ethanoyl ,-NR 5r 6-NH-halogenacyl, preferably-NH-tribromo-acetyl base or-NH-trifluoroacetyl group, and R 7the optional phenyl replaced, preferred phenyl.
A thirteenth aspect of the present invention relates to the compound of general formula 6 '
Wherein R 1be selected from the optional benzyl replaced, the optional diphenyl-methyl replaced, the trityl optionally replaced and the optional menaphthyl replaced, R 2the optional benzoyl replaced, and R 3be selected from acyl group and the H of optional replacement.
Accompanying drawing explanation
Hereinafter in further detail the present invention is described with reference to accompanying drawing, wherein:
The X-ray powder diffraction figure of the crystal LNnT that Fig. 1 display is prepared according to embodiment 34.
Comparison (the continuous curve: according to the diffractogram of crystal LNnT prepared by embodiment 34 of the X-ray powder diffraction figure of the crystal LNnT that Fig. 2 display is prepared according to 34 and EP-A-1405856; Vertical line: the data taking from EP-A-1405856).
The crystal LNnT's that Fig. 3 display is prepared according to embodiment 34 is solid-state 13c-NMR spectrogram.
Embodiment
This specification sheets in the whole text in, term " alkyl ", individually or when being connected with another kind of atom or group, refers to the straight or branched alkyl with 1-6 carbon atom, as methyl, ethyl, n-propyl, sec.-propyl, justbutyl, isobutyl-, sec-butyl, the tertiary butyl etc.
In this application, term " aryl " refers to that homoaromaticity group is as phenyl or naphthyl.Preferably, Aryl means phenyl.
In this manual, term " acyl group " represent R-C (=O)-, wherein R can be H, alkyl or aryl, as formyl radical, ethanoyl, propionyl, butyryl radicals, valeryl, benzoyl etc.Described alkyl and aromatic yl residue can be substituted.
For the object of this specification and claims, term " optional replacement " refers to that Targeting groups can be able to be maybe unsubstituted with substituting group.
For the object of this specification and claims, term " replacement " refers to that Targeting groups is replaced by the group usually changing the bulk chemical characteristic of chain or ring.Substituting group can be used to change molecule character as a whole, as the ability of stability, solubility and formation crystal.One skilled in the art will know that other suitable substituent with similar size and charge characteristic that can be used as alternatives in given situations.
More generally, with term " alkyl ", " aryl " and " acyl group " in conjunction with time, term " optional replacement " is intended to represent that Targeting groups can be selected from following group and replace one or many, preferred 1-5 time, more preferably 1-3 time: alkyl (only to aryl and aromatic acyl group), hydroxyl, alkoxyl group (i.e. alkyl-oxygen base), carboxyl, oxo (forming ketone or aldehyde functionality), alkoxy carbonyl, alkyl-carbonyl, formyl radical, aryl, aryloxycarbonyl, aryloxy, arylamino, aryl carbonyl, amino, list and dialkyl amido, formamyl, list and dialkyl-7-amino carbonyl, alkyl-carbonyl-amino, cyano group, alkanoyloxy, nitro, alkylthio and halogen (F, Cl, Br, I).
Statement " by the removable group of catalytic hydrogenation " refers to such group, the C-O key of described group become known for the catalytic amount palladium of hydrogenolysis, Raney nickel or another kind of suitably metal catalyst exist under by adding hydrogen and cracking, cause producing OH group.This type of group is known by the technical staff and is fully discussed herein [such as P.G.M.Wuts and T.W.Greene:ProtectiveGroupsinOrganicSynthesis (blocking group in organic synthesis), JohnWiley & Sons (2007)].Suitable group comprises benzyl, diphenyl methyl (diphenyl-methyl), 1-menaphthyl, 2-menaphthyl or trityl group (trityl); they can be selected from following one or more groups separately and optionally replace: alkyl, alkoxyl group, phenyl, amino, acyl amino, alkylamino, dialkyl amido, nitro, carboxyl, alkoxy carbonyl, formamyl, N-alkyl-carbamoyl, N, N-dialkyl carbamoyl, azido-, haloalkyl or halogen.Preferably, if existed, such is substituted on aromatic ring.Especially preferred protecting group is the benzyl that the one or more groups being selected from alkyl or halogen optionally replace.More preferably, protecting group is selected from unsubstituted benzyl, 4-chlorobenzyl and 4-methyl-benzyl.The advantage of these especially preferred and preferred protecting groups is: the by product of hydrogenolysis only has toluene or replaces toluene.Even if in several tons of scales, such by product also easily can remove via evaporation and/or extraction process from water-soluble product oligosaccharides.
Conjugated group-NR 5r 6in-NH-halogenacyl, term " halogenacyl " refers to formula C nh xx y-C (=O)-halogenacyl, wherein Integer n is 1,2 or 3, x+y=2n+1, and prerequisite is x < y, and X is F, Cl and Br, as dichloro-acetyl, tribromo-acetyl base, trifluoroacetyl group, seven fluorine butyryl radicalies etc.
The present invention represents a kind of business method being applicable to the extensive preparation of LNnT, namely typically with the LNnT of preparation at least 1kg in batch, as at least 5kg, or at least 50kg, or even at least 200kg, the LNnT of such as at least 1 ton.Successful strategy is the crystalline intermediate based on introducing the simple and reliable purification process of relevant permission.Crystallization or recrystallize are from separated from contaminants product and one of simple and the most cheap method obtaining pure substance.In addition, in product development, one or more crystalline modifications (polymorphic form) of solid are provided to be an important factor, because different crystalline forms differently affects the character of compound, such as thermodynamic stability, solubility, density, water absorbability, electrical property (as specific inductivity, specific conductivity), mechanical properties (as friability, hardness, breaking strength, elasticity), optical property (as color, transparency, refraction) etc.It expands the technical ability that can be used for improving the material of product performance that science personnel have.
The present invention provides the method for the preparation of Galp β 1-4GlcNAcp β 1-3Galp β 1-4Glc in first aspect, said method comprising the steps of:
A) donor characterized by general formula 5 is made
-NR 5r 6be selected from-NAc 2,-NH-halogenacyl, phthalimide, tetrachloro phthalimide, 2,3-diphenylmaleimide and 2,3-dimethylmaleimide,
X is selected from halogen ,-OC (=NH) CCl 3,-OAc ,-OBz and-SR 7, wherein R 7be selected from alkyl and the optional phenyl replaced,
With the receptor response of general formula 6
Wherein R 1by the removable group of catalytic hydrogenolysis,
R2 be the optional acyl group that replaces and
R3 is selected from acyl group or the H of optional replacement,
Thus produce the compound of general formula 4
Wherein R 1, R 2, R 3, R 4with-NR 5r 6as defined above,
B) by the converting compounds of general formula 4 be the compound of general formula 1
Wherein R 1as defined above,
C) compound crystal of general formula 1 is made, and
D) make the compound of general formula 1 through catalytic reduction subsequently.
About step a), synthesize in the compound of general formula 5 (donor) with the reaction of the compound (acceptor) of general formula 6 under glycosylation condition according to the LNnT derivative through adequately protecting of general formula 4.
The coupling of the lactose acceptor of general formula 6 and the lactose amino group donor of general formula 5 can be carried out causing required glycation product under the existence of activator (promotor or catalyzer) in the mixture of aprotic solvent or aprotic solvent.Between new glucosides, bonding is formed in the following manner: by 3 '-OH group nucleophilic substitution of the acceptor according to general formula 6 according to the leavings group X of the donor of general formula 5.Two other functional groups participated in the reactant of reaction need to shield by protecting group.In addition, the present inventor recognizes that regioselectivity glycosylation can R wherein 3be the general formula 6 of H acceptor on realize.In such dihydroxyl acceptor, the reactivity of 3 ' calm-OH and 4 ' axial-OH is different: calm OH group can serve as stronger nucleophile under glycosylation condition.Therefore, by carefully selection condition as the addition manner etc. of donor reactivity, solvent, temperature, promotor character, reactant/promotor, can driving a reaction to form bonding instead of 1-4 coupling between required 1-3 glucosides.Stereoselectivity especially be should be noted that.Stereochemical outcome may be subject to such as following Different factor impact: whether donor C-2 place exists participation group; the character of leavings group X; solvent effect; the character of the protecting group on donor and acceptor; the character of promotor or catalyzer; temperature, pressure, the steric interaction etc. between donor and acceptor.At glucose when amino or lactose aminoderivative, develop a series of glycosylated different head activation and it is available for carbohydrate chemistry those skilled in the art.These methods by summary and handbook by extensive discussions, such as Demchenko (Ed.): HandbookofChemicalGlycosylation (chemical glycosylation handbook), Wiley (2008).In order to embodiment, depend on X group, briefly mention some total considerations below.
Glycosyl halide (X refers to F, Cl, Br, I) is often used in glycosylation, and reason is its easy accessibility and gratifying reactivity.Typically, for nucleophilic substitution, different head halogenide follows the reactive order of F < Cl < Br < I.Glycosylation is usually by heavy metal ion, mainly mercury or silver, and Lewis acid promotes.
Glycosyl tri-chloroacetimidate (X=-OC (=NH) CCl 3) can easily by free different head OH being added to Trichloroacetonitrile to prepare under inorganic or organic base catalytic.In typical glycosylation, the Lewis acid of catalytic amount is as trimethyl silyl fluoroform sulphonate or BF 3-etherate promotes coupling.
Glycosyl acetate in glycosylation or benzoic ether (X represents-OAc or-OBz) first experience electrophilic activation, provide reactive intermediate, then use the process of nucleophilic OH acceptor.The Typical activators selected is that Bronsted acid is (as TsOH, HClO 4, thionamic acid), Lewis acid is (as ZnCl 2, SnCl 4, fluoroform sulphonate, BF 3-etherate, trityl perchlorate, AlCl 3, trifluoromethanesulfanhydride anhydride) and their mixture.
Sulphur glycosides (X represents alkylthio or thiophenyl) can by following activation: close sulphur promotor is as mercury (II) salt, Br 2, I 2, NBS, NIS, trifluoromethanesulfonic acid, fluoroform sulphonate, BF 3-etherate, trimethyl silyl fluoroform sulphonate, trifluoromethanesulfonic acid dimethyl-methylthio group sulfonium, phenylselanyl fluoroform sulphonate, perchloric acid two collidine iodine, tetrabutylammonium iodide or its mixture, in the condensation reaction, preferably by Br 2, NBS, NIS and fluoroform sulphonate activation.
In preferred embodiments, glycosyl donor is the compound of general formula 5, wherein R 4the optional acyl group replaced ,-NR 5r 6be-NH-halogenacyl and X is-SR 7, wherein R 7be selected from the optional alkyl replaced or the phenyl optionally replaced; More preferably R 7it is the optional phenyl replaced; Even more preferably R 4ethanoyl ,-NR 5r 6be selected from-NH-tribromo-acetyl base and-NH-trifluoroacetyl group, R 7be phenyl and-SR 7for β type.Glycosylation at aprotic solvent as chloroform, methylene dichloride, toluene, two in alkane, THF, acetonitrile or its mixture (preferred chloroform or methylene dichloride), at NIS, NBS, Br 2, trifluoromethanesulfonic acid, silver trifluoromethanesulfonate, BF 3carry out under the activation of-etherate or its mixture.
In a further preferred embodiment, glycosyl acceptor is the compound of general formula 6, wherein R 1the benzyl and R that optionally replace 3be selected from H and the optional benzoyl replaced; More preferably R 1benzyl, R 2the benzoyl optionally replaced by chlorine and R 3be selected from H and optionally by the benzoyl that chlorine replaces, and OR 1for β type.
The donor characterized by general formula 5 can be prepared by ordinary method known in the art.A kind of method can imply the galactosylation [Carbohydr.Res.2001 such as such as Sherman of shielded glycosamine sulphur glycosides; 336; 13]; or the galactosylation of the methylamino glucoside derivative [Tetrahedron (tetrahedron) 1987 such as such as Kochetkov; 43,3109].Another kind of approach is the derivatize based on the double bond to D-lactal six acetic ester by following two kinds of methods: exchange [the Can.J.Chem.1982 such as such as Lemieux by the nitrated rear reduction to 2-azido-of azido-and the protection to the amine formed subsequently and the nitrate-halogenide on anomeric carbon, 60, 63], or by after nitroso-group chlorination to the acetylize of the oxime formed and the diborane reduction [TetrahedronLett.1978 such as such as Ponpipom, 20, 1717], described two kinds of methods all cause producing shielded lactose amino halide.The functionalization of lactose amine is also (after O-acetylize, amine protection and different head acetic ester-halogen are changed mutually) that can expect.By forming sulphur glycosides, other the different heads activation that it is contemplated that from halogenide Carbohydr.Res.2001 such as [, 336,13] such as Sherman or from acetic ester TetrahedronLett.1988 such as [, 29,4759] such as Sato.The another kind of glycosyl donor often used is the tri-chloroacetimidate [TetrahedronLett.1988 such as such as Sato, 29,, and the amino fluorochemical of lactose (from corresponding glycosyl trinitride synthesis) is also disclosed [such as the application of donor 4759] deng Carbohydr.Res.1993,249,221].Above-mentioned literature examples is only the path illustrating that some is possible without limitation, and they can combine thus obtain the embodiment needed for general formula 5 characterizes by those skilled in the art.
The compound of general formula 6 can be obtained by following operation.By the eight-O-acetyl lactose commonly used or seven-O-acetyl lactose bromides, at Lewis acid (such as mercury salt, BF 3-etherate) activation under, utilize R 1oH can form corresponding lactoside.After going-O-acetylize (such as Zempl é n-goes protection, ammonia solution or alkaline hydrolysis); utilize the regioselectivity acetonization of Propanal dimethyl acetal in the presence of acid catalyst; the lactoside of 3 ', 4 '-protection can be obtained, then utilize R at typical condition 2-halogenide or (R 2) 2o (acid anhydrides) is by its acidylate.Acid can be utilized the hydrolysis of the derivative of gained thus remove isopropylidene, producing glycol (compound of general formula 6, wherein R 3oH), it utilizes derived from R 3the ortho ester process of OH.With acid catalyst, thus obtained cyclic ortho ester is rearranged into another compound of general formula 6 subsequently, wherein R 3that acyl group [is shown in the Carbohydr.Res.1985 such as such as Paulsen, 137,39; The ibid.1997 such as Lubineau, 305,501; And the reference wherein quoted] (scheme 2).
Step b in a first aspect of the present invention) in, the compound of general formula 4
Wherein R 1by the removable group of catalytic hydrogenolysis, R 2and R 4the optional acyl group replaced independently of one another, R 3be selected from acyl group or the H ,-NR of optional replacement 5r 6be selected from-NAc 2,-NH-halogenacyl, phthalimide, tetrachloro phthalimide, 2,3-diphenylmaleimide and 2,3-dimethylmaleimide) be converted into the compound of general formula 1, comprise the following steps:
Ba) base-catalyzed transesterification of the compound of general formula 4 removes protection or alkaline hydrolysis, wherein-NR 5r 6-NAc 2, thus produce the compound of general formula 1, or
Bb) base-catalyzed transesterification of the compound of general formula 4 goes protection, wherein-NR 5r 6be selected from-NH-halogenacyl, phthalimide, tetrachloro phthalimide, 2,3-diphenylmaleimide and 2,3-dimethylmaleimide, thus produce the compound of general formula 3
Wherein R 1with-NR 5r 6be as defined above, the compound of this general formula 3 through alkaline hydrolysis or ammonia solution, thus produces the compound of general formula 2
Wherein R 1be as defined above, the compound of this general formula 2 is converted into the compound of general formula 1, or
Bc) alkaline hydrolysis of the compound of general formula 4, wherein-NR 5r 6be selected from-NH-halogenacyl, 2,3-diphenylmaleimide and 2,3-dimethylmaleimide, thus produce the compound of general formula 2
Wherein R 1be as defined above, the compound of this general formula 2 is converted into the compound of general formula 1, or
Bd) alkaline hydrolysis of the compound of general formula 4, wherein-NR 5r 6be selected from phthalimide and tetrachloro phthalimide, then carry out ammonia solution, thus produce the compound of general formula 2
Wherein R 1be as defined above, the compound of this general formula 2 is converted into the compound of general formula 1, or
Be) the ammonia solution of the compound of general formula 4, wherein-NR 5r 6be selected from-NAc 2,-NH-halogenacyl, phthalimide, tetrachloro phthalimide, 2,3-diphenylmaleimide and 2,3-dimethylmaleimide, thus produce the compound of general formula 2
Wherein R 1be as defined above, the compound of this general formula 2 is converted into the compound of general formula 1.
Term " base-catalyzed transesterification go protection " refers to such reaction, wherein from the acyl protecting groups of hydroxyl at alcoholic solvent as in methyl alcohol, ethanol, propyl alcohol, the trimethyl carbinol etc., at alcoholate as NaOMe, NaOEt, KO tbu etc. are removed under existing at 20-100 DEG C of temperature.Alcohol and alcoholate should mate.The use of solubility promoter as toluene or dimethylbenzene may be useful to control the granularity of product, and avoids gel formation.With this understanding, only O-acyl group can be gone protection, or works as R 5and R 6when being all ethanoyl, one in acyl group is also removed thus produces and has the substituent compound of-NHAc.Under the de-protected condition of base-catalyzed transesterification ,-NH-halogenacyl and cyclic imide protecting group keep complete.In a preferred embodiment, the NaOMe (Zempl é n goes-O-acylations) of the catalytic amount in methyl alcohol is used in.
Term " alkaline hydrolysis " is often referred in water, alcohol or water-ORGANIC SOLVENT MIXTURES, in homogeneous phase or heterogeneous reaction condition, at the alkali catalyzed hydrolysis of the temperature of 0-100 DEG C.Selected alkali normally highly basic, such as LiOH, NaOH, KOH, Ba (OH) 2, K 2cO 3, deacidite, tetra-alkyl ammonium hydroxide etc.Described alkali also can use in form of an aqueous solutions.This condition affects O-acyl group, N-halogenacyl, 2,3-diphenylmaleimide and 2,3-dimethylmaleimide.If R 5and R 6be all ethanoyl, then one of acyl group is also removed.In a preferred embodiment, described alkali is NaOH and described solvent is methyl alcohol.
Term " ammonia solution " or based on N-acyl group transfer protection of going refer to, at the temperature of 20-120 DEG C, in water, alcohol or water-ORGANIC SOLVENT MIXTURES, utilize ammoniacal liquor, hydrazine, replacement hydrazine, quadrol or primary amine process.With this understanding, easily can remove all O-and N-protected acyl group, comprise cyclic imide.
According to another embodiment, the compound of the general formula 2 of acquisition is by N-acetylize.Selective N-acetylize under one or more hydroxyl exists is the reaction known and carries out the part general knowledge that such reaction is also those skilled in the art.Its relate to amine with little over the reaction about 0-35 DEG C (being with or without under adding alkali) of the diacetyl oxide measured or Acetyl Chloride 98Min. (≈ 1.5-3 equivalent).Utilize such as NaOH/MeOH or NaOMe/MeOH process, what finally formed crosses the compound that acetylize by product easily can be converted into required general formula 1.In another approach, the derivative according to general formula 2 is crossed acetylize, and namely free amine group and all free hydroxyl groups are all acetylation.Carry out reacting until want protected all groups to be acetylation belong to the ability of those skilled in the art.This compound diacetyl oxide or Acetyl Chloride 98Min., preferred diacetyl oxide, at alkali, there is lower process in preferred pyridine, triethylamine or H ü nig alkali, thus produces the group of the tetrose adequately protected of general formula 4, and it is characterized by general formula 4a
Wherein R 1as defined above.Then, cross acetyl derivatives 4a and go protection or alkaline hydrolysis (see above) through base-catalyzed transesterification, go-O-acetylating preferably through Zempl é n, thus produce the compound of general formula 1.
No matter once by taking which kind of approach above-mentioned to obtain the compound of general formula 1, they are separated with crystalline form.The present inventor recognizes that the compound of general formula 1 is crystalline substance.Because the compound of general formula 1 be in the process producing LNnT last intermediate and last go protection step not form by product actually under carry out, so the purity of their purity and target product LNnT is proportional.
When there is no crystal seed, from comprising one or more C 1-C 6the solvent of alcohol carries out crystallization.Term " C 1-C 6alcohol " refer to the alcohol with 1 to 6 carbon atom, i.e. methyl alcohol, ethanol, n-propyl alcohol, Virahol, the trimethyl carbinol, primary isoamyl alcohol etc.Preferred selection methyl alcohol or ethanol.More preferably, solvent systems can also comprise water.Water-content scope in the cumulative volume of solvent systems preferably can as many as 30v/v%, more preferably 15-25v/v%.
In a preferred embodiment, 1-O-phenyl LNnT crystallization from aqueous methanol or aqueous ethanol.
Steps d in first aspect present invention) in, the compound of general formula 1, through catalytic reduction, produces LNnT.
R 1the removal of group usually occurs in the mixture of protonic solvent or protonic solvent.Protonic solvent can be selected from by the following group formed: water, acetic acid or C 1-C 6alcohol.Also can adopt one or more protonic solvents and one or more can be partially or completely miscible with this protonic solvent appropriate aprotic organic solvent (as THF, two alkane, ethyl acetate, acetone etc.) mixture.Water, one or more C 1-C 6alcohol or water and one or more C 1-C 6the mixture of alcohol is preferably used as solvent systems.Also the suspension of solution containing the sugar derivatives of any concentration or sugar derivatives and solvent for use can be used.Reaction mixture is the temperature range of 10-100 DEG C, preferably in 20-70 DEG C of nitrogen atmosphere at 1-50bar in catalyzer lower stirring as palladium, Raney nickel or any other suitable metal catalyzer (preferred carbon supported palladium or palladium black) exist, complete until reach reaction.Based on the weight of sugar, catalyst metal concentration scope is generally 0.1% to 10%.Preferably, catalyst concn scope is 0.15% to 5%, more preferably 0.25% to 2.25%.When hydrogen produces from tetrahydrobenzene, cyclohexadiene, formic acid or ammonium formiate original position, also transfer hydrogenation can be carried out.The interpolation of organic or inorganic alkali/acid and/or alkalescence and/or acidic ion exchange resin also may be used for the kinetics improving hydrogenolysis.When in the substituted benzyl part that halogenic substituent is present in precursor, especially preferably use alkaline matter.Preferred organic bases includes but not limited to triethylamine, diisopropylethylamine, ammonia, ammonium carbamate, diethylamine etc.Preferred organic/inorganic acid includes but not limited to formic acid, acetic acid, propionic acid, Mono Chloro Acetic Acid, dichloro acetic acid, trifluoroacetic acid, HCl, HBr etc.Above-mentioned conditions permit is simple, convenient and accurately remove solvent, thus produces pure LNnT.Routine work program can be used from reaction mixture, to be separated LNnT with crystal, non-crystalline solid, jelly form or concentrated aqueous solutions.
In a preferred embodiment, 1-O-phenyl LNnT is made through catalytic hydrogenolysis to produce LNnT.Hydrogenation can in water or aqueous alcohol, preferably in water/methyl alcohol or water/alcohol mixture (alcohol content: 10-50v/v%) at 15-65 DEG C, carry out at preferred 60-65 DEG C.The concentration of raw material can change between 140-230g/l and the scope of catalyst concn can be 0.4% to 1.2% (the metal content weight based on sugar weight).
The LNnT purity as high in the aqueous solution/jelly all has by the LNnT of LNnT as noncrystalline/freeze-drying/spraying dry and liquid form of solid form provided by the invention, it is suitable for infant nutrition purposes, includes but not limited to infant formula (formulas), baby's cereal, clinical infant nutrition etc.Usually, the solid produced by method of the present invention and the LNnT of liquid form are suitable for the usual alimentary uses of baby, child, children, adult and the elderly.Also foodstuff additive, dietary supplements, alcohol and non-alcoholic beverage can be used as the component of soft drink, fruit juice, bottled water, grape wine, beer etc. by the LNnT of solid provided by the invention and liquid form.Also can as the therapeutical agent in extensive treatment use field by the LNnT of solid provided by the invention and liquid form, described treatment use field includes but not limited to pre-bacteriological protection and virus infection, avoids diarrhoea, strengthens immunity system and brain development etc.Also may be used to animal doctor's application by the LNnT of solid provided by the invention and liquid form, it includes but not limited to the infectious diseases resisting domestic animal.By LNnT provided by the invention also can be used as prepare polymkeric substance/be loaded with the key monomers of the product of polymkeric substance, described polymkeric substance/product that is loaded with polymkeric substance provides multivalence to combine for bacterium and virus.By applied chemistry and/or enzymatic means, also may be used for preparing other human milk oligosaccharides by LNnT provided by the invention, described chemistry and/or enzymatic means include but not limited to that the simple structure of the further extension of, core texture acid-group via the further fucosylation of N-acetyl lactose amination/N-acetyl neolactose amination etc., further saliva is modified.
Another aspect of the present invention relates to the novel crystal polymorphic form of LNnT.This novel crystal LNnT comprises based on the radiometric X-ray powder diffraction reflection of use CuK α, with lower angle place: at 20.32 ± 0.202 Θ angles, preferably 20.32 ± 0.20 and 19.10 ± 0.202 Θ angles, more preferably 20.32 ± 0.20, 19.10 ± 0.20 and 7.98 ± 0.202 Θ angles, even more preferably 20.32 ± 0.20, 19.10 ± 0.20, 7.98 ± 0.20 and 21.03 ± 0.202 Θ angles, most preferably 20.32 ± 0.20, 19.10 ± 0.20, 7.98 ± 0.20, 21.03 ± 0.20 and 20.95 ± 0.202 Θ angles, especially 20.32 ± 0.20, 19.10 ± 0.20, 7.98 ± 0.20, 21.03 ± 0.20, 20.95 ± 0.20 and 5.66 ± 0.202 Θ angles.List according to the peak of the XRPD figure of the crystal LNnT of embodiment 34 preparation shows in Table 1.
2Θ Relative intensity 2Θ Relative intensity
5.66 20 21.03 29
6.78 7 21.88 14
7.98 67 22.08 17
9.10 2 22.32 16
10.16 3 23.62 12
11.58 9 25.22 14
11.76 9 25.57 17
14.00 5 25.64 17
16.07 5 26.50 11
17.20 11 27.25 8
17.98 9 27.94 6
19.10 77 29.99 5
20.32 100 31.66 5
20.95 27 33.94 7
Table 1.
The new crystalline form of described LNnT can be considered to the pure form of α-anomer and β-one of anomeric different head mixture or even described anomer.If LNnT is separated as polycrystalline material, then it forms α-and β-anomeric mixture, wherein according to solid-state 13c-NMR measuring result, the relative β-anomer of α-anomer preponderates (ratio: be about 5: 2) (see Fig. 3).
The fusing point that Kuhn etc. [Chem.Ber.1962,95,513] report crystal LNnT is 214-218 DEG C (dec.).226-230 DEG C (dec) according to the fusing point of LNnT polymorphic form of the present invention.Significant difference between described fusing point shows that it relates to different polymorphic forms.
In European application EP-A-1405856, the crystal of LNnT is available from aqueous acetone solution, and the powder x-ray diffraction data that it records significantly are different from the powder x-ray diffraction data of the crystal of the application.The comparison display of described data in fig. 2.
Preferably, substantially organic solvent is not contained according to crystal LNnT of the present invention.Phrase " substantially not containing organic solvent " is intended to refer to that the content of organic solvent mostly is 1000ppm most, mostly preferably is most 800ppm, mostly more preferably is most 600ppm, mostly most preferably most be 400ppm and mostly especially most be 200ppm.
According to another preferred embodiment, the crystal LNnT that this application claims protection is pure substantially.Phrase " being pure substantially " is intended to refer to that described crystal LNnT (new polymorphic form) is containing the impurity being less than 10w/w%, preferably be less than the impurity of 5w/w%, more preferably less than the impurity of 1w/w%, most preferably be less than the impurity of 0.5w/w%, especially the impurity of 0.1w/w% is less than, wherein " impurity " refers to any physical individual being different from the crystal LNnT that the application describes, as noncrystal LNnT, different LNnT polymorphic forms, from the unreacted intermediate that the synthesis of LNnT stays, by product, degraded product, inorganic salt and/or be different from other pollutents of organic solvent.
The present invention also provides by from comprising one or more C 1-C 6the method of crystal LNnT is prepared in the solvent systems crystallization of alcohol.More preferably, this solvent systems can also contain water.The scope of the water-content in the cumulative volume of solvent systems can preferred as many as 40v/v%.This solvent mixture of 3-15 volume preferably can be used to carry out crystallization.
In typical crystallization, by take from removing catalyzer after the reaction mixture of hydrogenation or the LNnT of fresh preparation at water/methyl alcohol or at water/ethanol, solution (concentration: 140-180g/l) preferably in water/methyl alcohol (≈ 1: 1) mixture is warming to 50-60 DEG C, divides 2-4 part to add hot methanol (115-250% of the initial volume of as many as) in stirring wherein with being cooled to gradually at 35-45 DEG C.Crystallization can by adding kind of crystalline substance to cause.Then the warm suspension of gained is cooled to 0-8 DEG C carefully and optionally Keep agitation 2-5 hour.By the crystalline substance that filtering separation is formed.
In further embodiment, crystal LNnT according to the present invention is applicable to medicine and alimentary uses.Independent LNnT or with LNnT combine containing other N-acetyl lactosamines of human milk oligosaccharides and/or Fucose and/or sialic acid neonatal immune system enlighten by education and/or ripe in especially effectively, and for the secondary infection after virus infection (as influenza), there is preventive effect.LNnT is used to enhance prebiotics (as milk-acid bacteria (Lactobacillus) and bifidus bacillus (Bifidobacterium) species) in the formation of early stage bifid intestinal microbial group promoting baby as probiotics (prebiotic), in the formation risk reducing infantile allergy and/or asthma, the beneficial effect in the pathogen infection in such as suffering from diarrhoea of prevention and therapy baby and effect.
On the other hand, the invention provides pharmaceutical composition, described pharmaceutical composition comprises as the crystal LNnT of the present invention of activeconstituents and one or more pharmaceutical carriers, includes but not limited to additive, adjuvant, vehicle and thinner (water, gelatin, talcum, carbohydrate, starch, gum arabic, vegetable jelly, vegetables oil, polyalkylene glycol, seasonings, sanitas, stablizer, emulsifying agent, lubricant, tinting material, weighting agent, wetting agent etc.).Suitable carrier is described in the Remington ' sPharmaceuticalSciences (Standard reference works in this area) of latest edition.Formulation for administration comprises such as, tablet, pulvis, granula, pill, suspensoid, emulsifying agent, preserved material, capsule, syrup, injection, liquid, elixir, extractum and tincture.
In further embodiment, crystal LNnT according to the present invention is used to pharmaceutical compositions.Pharmaceutical composition can be produced by means of any usual mode as known in the art, such as, be described in the mode in the Remington ' sPharmaceuticalSciences (Standard reference works in this area) of latest edition.
In further embodiment, provide and comprise according to the nutritional formulation of crystal LNnT of the present invention as food, beverage or feed.This nutritional formulation also can comprise edible trace nutrient, VITAMIN and mineral substance.Whether the amount of such composition can be intended to for the baby of normal health, children, be grown up or have the special object (such as suffering from metabolism disorder) that needs and change according to said preparation.Trace nutrient comprises such as edible oil, fat or lipid acid (as cocounut oil, soya-bean oil, monoglyceride, triglyceride, palm olein, Trisun Oil R 80, fish oil, linolic acid, linolenic acid etc.), carbohydrate (as glucose, fructose, sucrose, maltodextrin, starch, hydrolysed corn starch etc.) and the albumen from casein, soybean, whey or skimming milk, or the hydrolysate of these albumen, and also can use the albumen (complete or hydrolysis) from other sources.VITAMIN can be selected from by the following group formed: vitamin A, B1, B2, B5, B6, B12, C, D, E, H, K, folic acid, inositol and nicotinic acid.Nutritional formula can comprise following mineral substance and trace elements: Ca, P, K, Na, Cl, Mg, Mn, Fe, Cu, Zn, Se, Cr or I.
In a preferred embodiment, described nutritional formulation is infant formula.Infant formula refers to such food, and it is designed for being in the special nutrition purposes of the baby of head 4-6 month period of life and itself meets the nutritional requirement of baby.It can comprise one or more probiotic bifidobacterium species, prebiotics as Nutriflora P and galactooligosacchari(es, from the albumen of casein, soybean, whey or skimming milk, sugar as required VITAMIN and mineral substance in lactose, sucrose, maltodextrin, starch or its mixture, lipid (such as palm olein, Trisun Oil R 80, Thistle oil) and diet.What infant formula comprised is 0.1-3.0g/100g formula food according to the total amount of crystal LNnT of the present invention.
In another preferred embodiment, described nutritional formulation can be comprise the dietary supplement ingredient according to crystal LNnT of the present invention.This dietary supplement ingredient can comprise one or more prebioticses, and its amount is for being enough to the effect in individuality needed for (preferably in children and adult) realization.This dietary supplement ingredient can also comprise VITAMIN, mineral substance, trace elements and other trace nutrients.This dietary supplement ingredient can be the form of such as tablet, capsule, lozenge or liquid.Described enriching substance can comprise and is selected from but is not limited to following conventional additives: tackiness agent, dressing, emulsifying agent, solubilizing agent, encapsulation agent, membrane-forming agent, sorbent material, carrier, weighting agent, dispersion agent, wetting agent, jelly (jellifyingagent), jelling agent etc.The per daily dose of LNnT is 0.1 to 3.0g.
According to a preferred embodiment, this dietary supplement ingredient is digestive health functional food because LNnT give to digestive health produce beneficial effect.Digestive health functional food is a kind of processed food, and the object of use is by strengthening using the crystal LNnT according to the present invention as physiological function composition or component of the forms such as tablet, capsule, pulvis and keep digestive health.Different terms such as dietary supplements, nutritive food, design food, healthy products also may be used for function as pronoun food.
In further embodiment, crystal LNnT according to the present invention is used to prepare nutritional formulation, and described nutritional formulation comprises food, beverage and feed, preferred infant formula, dietary supplement ingredient and digestive health functional food.This nutritional formulation can be prepared in any usual manner.
Think that the compound of general formula 1,2,3,4,5 and 6 is valuable synthetic intermediate for LNnT.The present inventor shockingly finds that the part in the compound of general formula 1,2,3,4,5 and 6 can obtain with crystalline form.Crystallization or recrystallize be from reaction mixture separated product, from separated from contaminants product and obtain pure substance the most simply and one of the most cheap method.Utilize the isolated or purified of crystallization to make whole technical process reliable and cost is effective, therefore compared with other programs, it has advantage and attractive.The present invention has immense commercial value in the scale operation of LNnT, provides highly purified intermediate, and this is irrealizable by any other known purification methods institute.Although other intermediates of a part do not demonstrate the ability of crystallization, but they can be prepared in the reaction that formed of less by product in clean, high yield, wherein routine operation (extraction, evaporation, precipitation etc.) program has been enough to obtain high purity product, and it is used in next step under not being further purified.
Therefore, the LNnT intermediate of valuable general formula 1 ' is provided
Wherein R ' 1be selected from the benzyl of replacement, optional diphenyl-methyl, the optional trityl replaced and the menaphthyl optionally replaced replaced.
It is stressed that the new derivatives characterized by general formula 1 ' can be considered independent chemical entity as α anomer or β anomer or or even α anomer and the anomeric different head mixture of β, preferred β anomer.The novel LNnT intermediate of general formula 1 ' can be characterized by crystalline solid, oily matter, jelly, precipitation amorphous material or Spray dried products.If crystal, the compound of general formula 1 ' can with anhydrous crystal forms existence or by existing with crystalline hydrated form in conjunction with one or several water moleculess in its crystalline structure.Similarly, the novel cpd characterized by general formula 1 ' can exist as being combined with in its crystalline structure the crystalline substance of part as organic molecule and/or ion.
In a preferred embodiment, R 1the benzyl replaced, preferred 4-chlorobenzyl or 4-methyl-benzyl.
Can by using chemistry/enzymatic means as known in the art for the preparation of LNnT self and other LNnT derivatives by the novel cpd of general formula 1 ' provided by the invention.The novel cpd of general formula 1 ' also can be used as the senior precursor/intermediate producing/prepare a large amount of human milk oligosaccharides.The novel cpd of general formula 1 ' also can be considered to the valuable intermediate for the synthesis of the complex oligosaccharide/glycoconjugate being suitable for treatment/alimentary uses.
Provide the LNnT intermediate of valuable general formula 2 '
Wherein R ' 1be selected from the benzyl of replacement, optional diphenyl-methyl, the optional trityl replaced and the menaphthyl optionally replaced replaced.
It is stressed that the new derivatives characterized by general formula 2 ' can be considered independent chemical entity as α anomer or β anomer or or even α anomer and the anomeric different head mixture of β, preferred β anomer.The novel LNnT intermediate of general formula 2 ' can be characterized by crystalline solid, oily matter, jelly, the amorphous material of precipitation or Spray dried products.If crystal, the compound of general formula 2 ' can with anhydrous crystal forms existence or by existing with crystalline hydrated form in conjunction with one or several water moleculess in its crystalline structure.Similarly, the novel cpd characterized by general formula 2 ' can exist as being combined with in its crystalline structure the crystalline substance of part as organic molecule and/or ion.
In a preferred embodiment, R 1the benzyl replaced, preferred 4-chlorobenzyl or 4-methyl-benzyl.
Can by using chemistry/enzymatic means as known in the art for the preparation of LNnT self and other LNnT derivatives by the new compound of general formula 2 ' provided by the invention.The novel cpd of general formula 2 ' also can be used as the senior precursor/intermediate producing/prepare a large amount of human milk oligosaccharides.The new compound of general formula 2 ' also can be considered to the valuable intermediate for the synthesis of the complex oligosaccharide/glycoconjugate being suitable for treatment/alimentary uses.
In addition, the compound of general formula 3 is provided
Wherein R 1be selected from the optional benzyl, optional diphenyl-methyl, the trityl optionally replaced and the optional menaphthyl replaced replaced that replace, and-NR 5r 6be selected from-NH-halogenacyl, phthalimide, tetrachloro phthalimide, 2,3-diphenylmaleimide and 2,3-dimethylmaleimide.
It is stressed that the new derivatives characterized by general formula 3 can be considered independent chemical entity as α anomer or β anomer or or even α anomer and the anomeric different head mixture of β, preferred β anomer.The new LNnT intermediate of general formula 3 can be characterized by crystalline solid, oily matter, jelly, the amorphous material of precipitation or Spray dried products.If crystal, the compound of general formula 3 can with anhydrous crystal forms existence or by existing with crystalline hydrated form in conjunction with one or several water moleculess in its crystalline structure.Similarly, the novel cpd characterized by general formula 3 can exist as being combined with in its crystalline structure the crystalline substance of part as organic molecule and/or ion.
In a preferred embodiment, R 1be selected from benzyl, 4-methyl-benzyl and 4-chlorobenzyl, preferred benzyl, and-NR 5r 6-NH-halogenacyl, preferably-NH-tribromo-acetyl base.
Can by using chemistry/enzymatic means as known in the art for the preparation of LNnT self and other LNnT derivatives by the novel cpd of general formula 3 provided by the invention.The novel cpd of general formula 3 also can be used as the senior precursor/intermediate producing/prepare a large amount of human milk oligosaccharides.The novel cpd of general formula 3 also can be considered to the valuable intermediate for the synthesis of the complex oligosaccharide/glycoconjugate being suitable for treatment/alimentary uses.
In addition, the compound of general formula 4 ' is provided
Wherein R 1be selected from the optional benzyl, optional diphenyl-methyl, the trityl optionally replaced and the optional menaphthyl replaced replaced that replace, and R 2and R 4the optional acyl group replaced independently of one another, R 3be selected from acyl group and the H ,-NR of optional replacement 5r 6be selected from-NAc 2,-NH-halogenacyl, tetrachloro phthalimide, 2,3-diphenylmaleimide and 2,3-dimethylmaleimide.
It is stressed that the new derivatives characterized by general formula 4 ' can be considered independent chemical entity as α anomer or β anomer or or even α anomer and the anomeric different head mixture of β, preferred β anomer.The novel LNnT intermediate of general formula 4 ' can be characterized by crystalline solid, oily matter, jelly, the amorphous material of precipitation or Spray dried products.If crystal, the compound of general formula 4 ' can with anhydrous crystal forms existence or by existing with crystalline hydrated form in conjunction with one or several water moleculess in its crystalline structure.Similarly, the new compound characterized by general formula 4 ' can exist as being combined with in its crystalline structure the crystalline substance of part as organic molecule and/or ion.
In a preferred embodiment, R 1be selected from benzyl, 4-methyl-benzyl and 4-chlorobenzyl, preferred benzyl, R 2optionally by the benzoyl that chlorine replaces, R 3the optional benzoyl replaced, preferred benzoyl, R 4be ethanoyl and-NR 5r 6-NH-halogenacyl, preferably-NH-tribromo-acetyl base.
Can by using chemistry/enzymatic means as known in the art for the preparation of LNnT self and other LNnT derivatives by the novel cpd of general formula 4 ' provided by the invention.The novel cpd of general formula 4 ' also can be used as the senior precursor/intermediate producing/prepare a large amount of human milk oligosaccharides.The novel cpd of general formula 4 ' also can be considered to the valuable intermediate for the synthesis of the complex oligosaccharide/glycoconjugate being suitable for treatment/alimentary uses.
In addition, the compound of general formula 4a is provided
Wherein R 1be selected from the optional benzyl, optional diphenyl-methyl, the trityl optionally replaced and the optional menaphthyl replaced replaced that replace.
It is stressed that the new derivatives characterized by general formula 4a can be considered independent chemical entity as α anomer or β anomer or or even α anomer and the anomeric different head mixture of β, preferred β anomer.The novel LNnT intermediate of general formula 4a can be characterized by crystalline solid, oily matter, jelly, the amorphous material of precipitation or Spray dried products.If crystal, the compound of general formula 4a can with anhydrous crystal forms existence or by existing with crystalline hydrated form in conjunction with one or several water moleculess in its crystalline structure.Similarly, the new compound characterized by general formula 4a can exist as being combined with in its crystalline structure the crystalline substance of part as organic molecule and/or ion.
In a preferred embodiment, R 1be selected from benzyl, 4-methyl-benzyl and 4-chlorobenzyl, preferred benzyl.
The novel cpd of general formula 4a provided by the invention can by using chemistry/enzyme method as known in the art for the preparation of LNnT self (especially when the selective N-acetylize of the compound of general formula 2 is not effective) and other LNnT derivatives.The novel cpd of general formula 4a also can be used as the senior precursor/intermediate producing/prepare a large amount of human milk oligosaccharides.The novel cpd of general formula 4a also can be considered to the valuable intermediate for the synthesis of the complex oligosaccharide/glycoconjugate being suitable for treatment/alimentary uses.
Another aspect of the present invention relates to the novel crystal lactose amino group donor characterized by general formula 5 '
Wherein R 4the optional acyl group replaced ,-NR 5r 6-NH-halogenacyl, and R 7it is the optional phenyl replaced.
It is stressed that the new derivatives characterized by general formula 5 ' can be considered independent chemical entity as α anomer or β anomer or or even α anomer and the anomeric different head mixture of β, preferred β anomer.The new LNnT intermediate of general formula 5 ' can be characterized by crystalline solid, oil, slurries, the amorphous substance of precipitation or spray-dired product.If crystal, the compound of general formula 5 ' can with anhydrous crystal forms existence or by existing with crystalline hydrated form in conjunction with one or several water moleculess in its crystalline structure.Similarly, the novel cpd characterized by general formula 5 ' can exist as being combined with in its crystalline structure the crystalline substance of part as organic molecule and/or ion.
In a preferred embodiment, R 4ethanoyl ,-NR 5r 6-NH-tribromo-acetyl base or-NH-trifluoroacetyl group, R 7be phenyl and-SR 7for β type.
The compound of general formula 5 ' is stable, can standing storage and remarkable decomposition does not occur, and easily can be activated and demonstrate excellent beta selective in glycosylation.Since it is known other beta selective lactose amino group donor are not solids and/or stable, thus in lactose ammoxidation, there is significantly favourable applicability according to the compound of the general formula 5 ' of the application and therefore represent wherein containing the oligosaccharides of lactose amine as the valuable donor instrument in the synthesis (especially extensive or plant-scale) of target.
The compound of general formula 5 ' can be prepared as follows: lactose amine hydrochlorate is at Ac 2be acetylation in O/HBr/AcOH, thus produce 1,3,6,2 ', 3 ', 4 ' 6 '-seven-O-ethanoyl-lactose amine hydrochlorates, it turns to-NH-halogenacyl derivative with halogenation halogenacyl or acid anhydrides N-acyl group.Utilize HBr/AcOH to form bromo sugar, then utilize R 7sH thiophenol derivative carries out thiolysis, and easily obtains the compound of general formula 5 '.By the transesterification reaction of base catalysis, then follow-up acylations, can become other suitable acyl groups by ethanoyl.
Therefore, the invention provides the compound of valuable general formula 6 '
Wherein R 1be selected from the optional benzyl, optional diphenyl-methyl, the trityl optionally replaced and the optional menaphthyl replaced replaced that replace, R 2the optional benzoyl replaced, and R 3be selected from benzoyl and the H of optional replacement.
Preferred embodiment contains the compound of such general formula 6 ', wherein R 1the optional benzyl replaced, preferred benzyl, 4-methyl-benzyl or 4-chlorobenzyl, R 2the optional benzoyl replaced, preferred benzoyl or 4-chlorobenzene formacyl, and R 3benzoyl or H.
The present inventor recognizes, when the compound of general formula 6 serves as glycosyl acceptor in reaction of guanosine, as R 3ethanoyl be inconvenient protecting group.Under coupling condition, always acetyl group migration occur thus produce the mixture comprising the complexity of the material with similar physical property, these compounds only can be separated by technology such as chromatography that is tediously long and/or complicated and/or effort.The acyl protecting groups not being tending towards or not more being tending towards the more volume of moving is selected to cause producing following acceptor; it almost forms coupled product exclusively in glycosidation; make the processing sequence of required compound and sepn process simpler, fast, effectively and cost is effective; such as by crystallization, this one of pays close attention to the most in scale operation or commercial run.
In the following process be described exemplary, other features of the present invention will become obvious, and these exemplary are used for illustrating the present invention and being not used in restriction the present invention.
Embodiment
Embodiment 1
By the suspension of 10g benzyl β-D-lactoside in acetone (50ml), Propanal dimethyl acetal (3.5ml) and TMSCl (7ml) at stirring at room temperature 5h.Mixture ethyl acetate (50ml) dilution, filter, and filter cake ethyl acetate (2x30ml) is washed.Wet filter cake is dissolved in pyridine (36ml) and dry DCM (50ml), and slowly adds 4-chloro-benzoyl chloride (22ml) with holding temperature at 40-45 DEG C.After stirred overnight, add methyl alcohol (10ml) and DCM (10ml) and carry out extracting operation (2x1MHCl, 1x water, the saturated NaHCO of 1x 3).The organic phase of merging is concentrated and pours into vigorous stirring in 50ml Virahol as thick jelly.Filtering solids, also dry by washed with isopropyl alcohol, thus produce 20.0g benzyl 2,3,6,2 ', 6 '-five-O-(4-chlorobenzene formacyl)-3 ', 4 '-two-O-isopropylidene-β-D-lactoside (72%).[α] D＝+58.4°(c＝1DCM)，Mp：184℃。
Embodiment 2
By 10g benzyl 2,3,6,2 ', 6 '-five-O-(4-chlorobenzene formacyl)-3 ', 4 '-two-O-isopropylidene-β-D-lactoside is dissolved in DCM (20ml), acetonitrile (2ml) and 50%HClO 4(1ml) in, and by this mixture at stirring at room temperature 30min.Use saturated NaHCO 3(2x10ml) this solution is extracted, dry, filter and concentrate.The material of gained is dissolved in again in ethyl acetate (10ml) also with hexane (50ml) dilution.By this suspension at stirring at room temperature 30min, and filter, thus produce benzyl 2,3,6,2 ', 6 '-five-O-(the 4-chlorobenzoyl)-β-D-lactoside of 5.4g as white crystal.[α] D＝+58.65°(c＝1DCM)，Mp：200-201℃。
Embodiment 3
Propionyl chloride is utilized to prepare similarly according to embodiment 1 and 2: benzyl 2,3,6,2 ', 6 '-five-O-propionyl-β-D-lactoside.
1hNMR (CDCl 3, 300MHz) and δ: 7.346-7.227 (5H, Ph), 5.140 (1H, dd, J=9.1,9.7, H-3), 4.980 (1H, dd, J=7.9,9.7, H-2), 4.881 (1H, dd, J=7.99.7, H-2 '), 4.838 (1H, d, J=12.3,1/2C h 2ph), 4.570 (1H, d, J=12.3,1/2C h 2ph), 4.495 (1H, dd, J=1.8,11.7, H-6aorH-6a '), 4.343-4.282 (2H, unresolved, H-6a or H-6a ', H-1), 4.217-4.155 (2H, unresolved, H-6b, H-6b '), 3.821 (1H, d, J=3.4, H-4 '), 3.743 (1H, dd, J=9.8,9.8, H-4), and 3.606-3.537 (3H, unresolved, H-3 ', H-5, H-5 '), 2.438-2.194 (10H, m, 5 × C h 2cH 3), 1.263-1.036 (15H, m, 5 × CH 2c h 3).
13CNMR(CDCl 3，75.4MHz)δ：174.7，174.3，173.9，173.8，172.9，171.2(6×CO)，136.6-127.9(Ph)，100.6(C-1′)，99.1(C-1)，75.9(C-4)，73.3，72.9，72.7，72.3，72.2(C-2′，C-3，C-3′，C-5，C-5′)，71.2(C-2)，70.6( CH 2Ph)，68.4(C-4′)，62.2，62.0(C-6，C-6′)。
Embodiment 4
Benzoyl chloride is utilized to prepare similarly according to embodiment 1 and 2: benzyl 2,3,6,2 ', 6 '-five-O-benzoyl-β-D-lactoside.
1HNMR。(CDCl 3) δ: 8.20-7.10 (m, 30H, aromatics), 5.55 (dd, 1H, J 2,39.82Hz, J 3,49.82Hz, H-3), 5.49 (dd, 1H, J 1,29.78Hz, H-2), 5.35 (dd, 1H, J 1 ', 2 '7.85Hz, J 2 ', 3 '9.64Hz, H-2 '), 4.81 and 4.57 (ABq, 2H, J gem12.59Hz ,-C h 2ph), 4.62 (d, 1H, H-1), 4.57 (d, 1H, H-1 '), 4.52 (m, 2H, H-6), 4.12 (m, 1H, H-4), 3.80 (m, 1H, H-4 '), (3.75 m, 1H, H-5), (3.70 m, 1H, H-3 ').
1 3cNMR (CDCl 3) δ: 166.51,166.31,166.18,166.16 and 165.45 (5 × CO), 101.23 (C-1 '), 99.09 (C-1), 76.49 (C-4), 73.81 (C-2 '), 73.26,73.22,73.19 and 72.87 (C-3, C-5, C-3 ' and C-5 '), 72.80 (C-2), 71.77 (- ch 2ph), 68.81 (C-4 '), 62.85 and 61.99 (C-6 and C-6 ').
Embodiment 5
Prepare similarly from 4-methyl-benzyl β-D-lactoside according to embodiment 1 and 2: 4-methyl-benzyl 2,3,6,2 ', 6 '-five-O-(4-chlorobenzene formacyl)-β-D-lactoside.
1HNMR(CDCl 3)：7.8，7.4，7.2，7.0(m)，5.4(m，3H)，4.76(d，1H)，4.5(m，5H)，4.1(dd，1H)，4.0(m，2H)，3.84(m，1H)，3.72(m，2H)，3.6(m，2H)，3.48(m，1H)，3.3(d，1H)。
13CNMR(CDCl 3)：165.3，165.2，165.1，164.3，139.9，139.8，137.9，133.2，131.3，131.1，130.9，130.9，129.1，129.0，128.9，128.8，128.7，128.1，127.9，127.8，127.7，127.5，127.4，101.1，98.3，76.4，73.6，73.4，72.9，72.6，72.3，71.7，70.4，68.6，62.9，62.0，60.4，21.1，14.2。
Embodiment 6
To 116.6g according in the mixture of the compound of embodiment 2 in toluene (600ml), add former phenylformic acid trimethyl (120ml) and amphene sulfonic acid (4g).By mixture at room temperature vigorous stirring 3h, then add 80% acetic acid (160ml).After the stirring of another hour, the two-phase mixture obtained is separated, dilutes organic phase with toluene (600ml), with water (800ml) and saturated NaHCO 3(2x600ml) wash, dry, filter and evaporate.The oily matter of gained to be added dropwise in 600ml heptane and to add crystal seed.Filtered by white crystalline Compound, washing is also dry, thus produces 110.3g acceptor (compound of general formula 6, wherein R 1=Bn, R 2=4-chlorobenzene formacyl, R 3=benzoyl, OR 1for β type).[α] D＝+17.13°(c＝1DCM)，Mp156-157℃。
Embodiment 7
Prepare like the compounds of embodiment 4: the compound of general formula 6, wherein R 1=Bn, R 2=R 3=benzoyl, OR 1for β type.
1HNMR。(CDCl 3) δ: 8.20-7.00 (m, 35H, aromatics), 5.67 (dd, 1H, J 2,39.77Hz, J 3,49.11Hz, H-3), 5.55 (dd, 1H, J 1,27.81Hz, H-2), 5.47 (dd, 1H, J 3 ', 4 '3.45Hz, J 4 ', 5 '< 1Hz, H-4 '), 5.30 (dd, 1H, J 1 ', 2 '7.82Hz, J 2 ', 3 '9.97Hz, H-2 '), 4.85 and 4.60 (ABq, 2H, J gem12.59Hz ,-C h 2ph), 4.72 (d, 1H, H-1), 4.70 (d, 1H, H-1 '), 4.60 (m, 2H, H-6), 4.21 (m, 1H, H-4), 3.93 (m, 1H, H-3 '), 3.82 (m, 1H, H-5), 3.75-3.45 (m, 3H, H-5 ' and H-6 ') 2.70 (d, 1H, J 3 ', OH6.68Hz, 3 '-OH).
13cNMR (CDCl 3) δ: 166.67,166.26,166.03,165.90,165.68 and 165.41 (6 × CO), 100.79 (C-1 '), 99.35 (C-1), 76.16 (C-4), 73.89 (C-2 '), 73.26 (C-5), 72.96 (C-3), 72.11 (C-3 '), 71.79 (C-2), 71.76 (C-5 '), 70.73 (- ch 2ph), 70.22 (C-4 '), 62.87 and 61.68 (C-6 and C-6 ').
Embodiment 8
Prepare like the compounds of embodiment 5: the compound of general formula 6, wherein R 1=4-methyl-benzyl, R 2=4-chlorobenzene formacyl, R 3=benzoyl, OR 1for β type.
1hNMR (CDCl 3): 7.92-7.0 (m, aromatics H), 5.6 (dd, 1H), 5.5 (d, 1H), 5.41 (dd, 1H), 5.3 (dd, 1H), 4.8 (d, 1H), 4.68 (m, 2H), 4.52 (m, 2H), 4.08 (m), 4.0 (m, 2H), 3.78 (m, 3H), 3.6 (m, 1H).
13cNMR (CDCl 3): 165.7,165.2,165.1,164.7,164.5,164.3 (Bz and ClBz carbonyl), 140.1,140.02,139.9,139.7,139.6,137.8,133.7,133.1,131.2-127.2 aromatics carbon, 100.6,98.6 (anomeric carbons), 75.9,73.6,73.1,72.9,71.7,71.4,71.3,70.5,69.9,62.9,61.7,45.9,42.7,42.4,14.1.
Embodiment 9
According to embodiment 1,2 and 6, prepare similarly from 4-chlorobenzyl β-D-lactoside: the compound of general formula 6, wherein R 1=4-chlorobenzyl, R 2=4-chlorobenzene formacyl, R 3=benzoyl, OR 1for β type.
1hNMR (CDCl 3): 7.89-7.0 (m, aromatics H), 5.63 (dd, 1H), 5.5 (d, 1H), 5.42 (dd, 1H), 5.28 (dd, 1H), 4.78 (d, 1H), 4.68 (dd, 1H), 4.54 (m, 3H), 4.1 (m, 2H), 3.98 (m, 1H), 3.8 (m, 2H), 3.6 (m, 2H).
13CNMR(CDCl 3)：165.8，165.2，164.8，164.5，164。3，140.2，140.0，139.8，134.9，133.9，133.8。131.2，131.1，130.9，130.9，130.8，129.9，129.0，128.9，128.8，128.7，128.6，128.5，127.7，127.4，127.3，100.6，99.2，75.8，73.6，73.0，71.8，71.5，71.4，70.0，69.9，62.8，61.6，57.9，56.1，48.1，46.0，42.7，42.5。
Embodiment 10
At 0 DEG C, 10g lactose amine hydrochlorate is dissolved in Ac 2the 30%HBr/AcOH of 10ml is added in O (50ml).Allow reaction mixture to be warming up to room temperature, stir 10h and pour in 200ml t-butyl methyl ether (MTBE).Filtering-depositing, also dry with MTBE washing, thus obtain 1,3,6,2 ', 3 ', 4 ' 6 '-seven-O-ethanoyl-lactose amine hydrochlorates of 12.4g as light brown powder.1HNMR (CDCl3) δ: 5.57 and 5.22 (2m, 1H, H-1 α and β), 5.57,5.29,4.98 and 4.89 (4m, each 1H, H-3, H-2 ', H-3 ' and H-4 '), 4.41 (m, 1H, H-1 '), 4.37 and 4.06 (2m, 1H and 3H, H-6 and H-6 '), 3.83,3.82 and 3.73 (3m, each 1H, H-4, H-5 and H-5 '), 3.40 (m, 1H, H-2), 2.10,2.10,2.06,2.04,1.98,1.90 and 1.89 (7s, each 3H, 7 ×-OAc).13CNMR (CDCl3) δ: 100.87 (C-1 '), 92.91 and 88.93 (C-1 α and β), 75.64,73.03 and 71.01 (C-4, C-5 and C-5 '), 61.72 and 60.72 (C-6 and C-6 '), 54.08 (C-2).
Embodiment 11
By 16.4g1,3,6,2 ', 3 ', 4 ' 6 '-seven-O-ethanoyl-lactose amine hydrochlorates to be dissolved in DCM (30ml) and to be cooled to 0 DEG C.First add 2.95ml trichoroacetic chloride, then slowly add 7.6ml triethylamine and continue to stir 30min at 0 DEG C.This reaction mixture is diluted, with water (2x20ml) and salt solution (20ml) washing, at Na with DCM (10ml) 2sO 4upper drying is also filtered.At 0 DEG C, by this solution and Ac 2mixture instillation DCM (15ml) of O (100 μ l), HBr/AcOH (15ml) and Ac 2in the solution of O (1ml) and uniform temp keep 30min.This reaction mixture is diluted, with cold water (3x20ml) and cold saturated NaHCO with DCM (10ml) 3(20ml) wash.100ml semi-saturation Na is added in organic phase 2cO 3solution, 4.3ml thiophenol and 100mg 4-butyl ammonium hydrogen sulfate by this two-phase mixture at stirring at room temperature 30min.After isolation, wash organic phase with salt solution (2x20ml), and after adding ethyl acetate (50ml), filter the crystal (6.3g) formed.By adding MTBE (50ml), from dense mother liquor, be settled out 3.2g crystal further.Merge yield: 9.5g product (compound of general formula 5, wherein R 4=ethanoyl ,-NR 5r 6=-NH-tribromo-acetyl base, X=β-SPh). 1HNMR。(CDCl 3) δ: 7.50 and 7.30 (2m, 5H, aromatics), 7.08 (d, 1H, J nH, 29.37Hz, NH), 5.33 (dd, 1H, J 3 ', 4 '3.19Hz, J 4 ', 5 '0.65Hz, H-4 '), 5.21 (dd, 1H, J 2,38.34Hz, J 3,410.12Hz, H-3), 5.08 (dd, 1H, J 1 ', 2 '7.83Hz, J 2 ', 3 '10.47Hz, H-2 '), 4.94 (dd, 1H, H-3 '), 4.75 (d, 1H, J 1,210.34Hz, H-1), 4.55 and 4.09 (2m, 4H, H-6 and H-6 '), 4.47 (d, 1H, H-1 '), 4.00 (m, 1H, H-2), 3.87,3.78 and 3.67 (3m, each 1H, H-4, H-5 and H-5 '), 2.16,2.11,2.10,2.04,2.03 and 1.96 (6s, each 3H, 6 ×-OAc). 13cNMR (CDCl 3) δ: 100.47 (C-1 '), 85.1 (C-1), 75.99,75.41 and 69.97 (C-4, C-5 and C-5 '), 72.50 (C-3), 70.17 (C-3 '), 68.42 (C-2 '), 65.87 (C-4 '), 61.43 and 60.26 (C-6 and C-6 '), 53.66 (C-2), 21.35,21.11,21.11,20.98,20.87 and 20.75 (6 × OAc).Mp.：247-249℃，[α] D＝-13，9°(c＝0，58CHCl 3)。
Embodiment 12
Prepare similarly according to embodiment 11: the compound of general formula 5, wherein R 4=ethanoyl ,-NR 5r 6=-NH-dichloro-acetyl, X=β-SPh.
1hNMR (CDCl 3) δ: 7.50 and 7.30 (2m, 5H, aromatics), 7.08 (d, 1H, J nH, 29.37Hz, NH), 5.33 (dd, 1H, J 3 ', 4 '3.19Hz, J 4 ', 5 '0.65Hz, H-4 '), 5.21 (dd, 1H, J 2,38.34Hz, J 3,410.12Hz, H-3), 5.08 (dd, 1H, J 1 ', 2 '7.83Hz, J 2 ', 3 '10.47Hz, H-2 '), 4.94 (dd, 1H, H-3 '), 4.75 (d, 1H, J 1,210.34Hz, H-1), 4.55 and 4.09 (2m, 4H, H-6 and H-6 '), 4.47 (d, 1H, H-1 '), 4.00 (m, 1H, H-2), 3.87,3.78 and 3.67 (3m, each 1H, H-4, H-5 and H-5 '), 2.16,2.11,2.10,2.04,2.03 and 1.96 (6s, each 3H, 6 ×-OAc).
13cNMR (CDCl 3) δ: 100.47 (C-1 '), 85.1 (C-1), 75.99,75.41 and 69.97 (C-4, C-5 and C-5 '), 72.50 (C-3), 70.17 (C-3 '), 68.42 (C-2 '), 65.87 (C-4 '), 61.43 and 60.26 (C-6 and C-6 '), 53.66 (C-2), 21.35,21.11,21.11,20.98,20.87 and 20.75 (6 × OAc).
Embodiment 13
Prepare similarly according to embodiment 11: the compound of general formula 5, wherein R 4=ethanoyl ,-NR 5r 6=-NH-trifluoroacetyl group, X=β-SPh.
1hNMR (CDCl 3) δ: 7.79 (d, 1H, J nH, 29.74Hz, NH), 7.45 and 7.29 (2m, 5H, aromatics), 5.35 (dd, 1H, J 3 ', 4 '3.10Hz, J 4 ', 5 '< 1Hz, H-4 '), 5.24 (m, 1H, H-3), 5.05 (dd, 1H, J 1 ', 2 '7.59Hz, J 2 ', 3 '10.44Hz, H-2 '), 4.96 (dd, 1H, H-3 '), 4.82 (d, 1H, J 1,210.35Hz, H-1), 4.46 (d, 1H, H-1 '), 4.65 and 4.10-3.88 (m, 7H, H-2, H-5, H-6, H-5 ' and H-6 '), 3.76 (m, 1H, H-4), 2.11,2.06,2.05,2.04,2.03 and 1.99 (6s, each 3H, 6 ×-OAc).
13CNMR (CDCl3) δ: 171.67,170.63,170.56,170.41,170.41 and 169.48 (6 × OAc), (101.64 C-1 '), 84.44 (C-1), 76.78 (C-4), 76.25 (C-5), 71.41 (C-3), 71.09 and 70.84 (C-3 ' and C-5 '), 69.22 (C-2 '), 66.70 (C-4 '), 62.06 and 60.87 (C-6 and C-6 '), 52.72 (C-2).
Embodiment 14
Prepare similarly according to embodiment 11: the compound of general formula 5, wherein R 4=4-chlorobenzene formacyl ,-NR 5r 6=-NH-dichloro-acetyl, X=β-SPh.
1hNMR (CDCl 3) δ: 7.92-7.05 (m, 29H, aromatics), 6.65 (d, 1H, J nH, 29.45Hz, NH), 5.74 (s, 1H ,-CHCl 2), 5.65 (dd, 1H, J 3 ', 4 '3.39Hz, J 4 ', 5 '< 1Hz, H-4 '), 5.55 (dd, 1H, J 1 ', 2 '7.89Hz, J 2 ', 3 '10.34Hz, H-2 '), 5.53 (dd, 1H, J 2,39.01Hz, J 3,410.24Hz, H-3), 5.32 (dd, 1H, H-3 '), 4.80 (d, 1H, J 1,210.31Hz, H-1), 4.78 (d, 1H, H-1 '), 4.52 and 4.35 (m, 2H, H-6), 4.01 (m, 1H, H-2), 3.91 (m, 1H, H-4), 3.90 (m, 3H, H-5 ' and H-6 '), 3.67 (m, 1H, H-5).
13cNMR (CDCl 3) δ: 165.42,165.16,164.89,164.74,164.37,164.37 and 164.25 (7 × CO), 100.87 (C-1 '), 86.27 (C-1), 76.98 (C-5), 75.99 (C-4), 73.99 (C-3), 71.67 (C-3 '), 71.01 (C-5 '), 70.12 (C-2 '), 67.69 (C-4 '), 66.36 (-CHCl 2), 62.71 and 61.05 (C-6 and C-6 '), 53.42 (C-2).
Embodiment 15
Prepare similarly according to embodiment 11: the compound of general formula 5, wherein R 4=benzoyl ,-NR 5r 6=-NH-dichloro-acetyl, X=β-SPh.
1hNMR (CDCl 3) δ: 8.18-7.08 (m, 35H, aromatics), 6.84 (d, 1H, J nH, 29.50Hz, NH), 5.82 (s, 1H ,-CHCl 2), 5.77 (dd, 1H, J 3 ', 4 '3.40Hz, J 4 ', 5 '< 1Hz, H-4 '), 5.69 (dd, 1H, J 1 ', 2 '7.88Hz, J 2 ', 3 '10.36Hz, H-2 '), 5.53 (dd, 1H, J 2,39.05Hz, J 3,410.27Hz, H-3), 5.42 (dd, 1H, H-3 '), 4.91 (d, 1H, J 1,210.21Hz, H-1), 4.91 (d, 1H, H-1 '), 4.65 and 4.44 (m, 2H, H-6), 4.14 (m, 1H, H-2), 4.05 (m, 1H, H-4), 3.83 (m, 3H, H-5 ' and H-6 '), 3.73 (m, 1H, H-5).
13cNMR (CDCl 3) δ: 166.51,165.97,165.84,165.67,165.45,165.05 and 164.39 (7 × CO), 101.30 (C-1 '), 86.63 (C-1), 76.86 (C-5), 75.85 (C-4), 73.91 (C-3), 71.90 (C-3 '), 71.65 (C-5 '), 70.11 (C-2 '), 67.64 (C-4 '), 66.25 (-CHCl 2), 62.79 and 61.28 (C-6 and C-6 '), 53.72 (C-2).
Embodiment 16
Amino for 3.0g methyl thio lactoside to be dissolved in 10mlDMF and to add 100 μ l triethylamines and 6.0ml methyl trichloroacetate in this solution.By this reaction mixture stirred overnight at room temperature, be then concentrated into dry, enriched material be dissolved in methyl alcohol (10ml), cool and add NaOMe until pH reaches 9.Stirred by this mixture and spend the night, evaporation, then adds pyridine (20ml) and Ac 2o (10ml).After 6h, this mixture concentrated and uses hexane-acetone 6: 4 to carry out chromatographic separation, thus obtaining canescence foam (compound of general formula 5, wherein R 4=ethanoyl ,-NR 5r 6=-NH-tribromo-acetyl base, X=SMe).For characterizing this anomer, stratographic analysis is carried out to analytic sample.α anomer: 1hNMR (CDCl 3) δ: 7.05 (d, 1H, J nH, 27.05Hz, NH), 5.30 (m, 2H, J 1,25.38Hz, H-1 and H-4 '), 5.13 (dd, 1H, J 2,310.99Hz, J 3,48.81Hz, H-3), 5.07 (dd, 1H, J 1 ', 2 '7.83Hz, J 2 ', 3 '10.42Hz, H-2 '), 4.92 (dd, 1H, J 3 ', 4 '3.37Hz, H-3 '), 4.49 (d, 1H, H-1 '), 4.38-4.13 (m, 2H, H-6 '), 4.26 (m, 1H, H-2), 4.21 (m, 1H, H-5), 4.03 (m, 2H, H-6), 3.85 (m, 1H, H-5 '), 3.76 (m, 1H, H-4), 2.09,2.09,2.07,2.02,2.02,2.01 and 1.99 (7 × s, each 3H, 6 × OAc and SMe). 13cNMR (CDCl 3) δ: 171.14, 170.28, 170.28, 170.01, 169.91, 169.03 with 161.75 (7 × CO), (100.96 C-1 '), 91.67 (TCA), 83.98 (C-1), 75.89 (C-4), 70.76 (C-3 '), 70.75 (C-3), 70.43 (C-5 '), 68.98 (C-5), 68.91 (C-2 ') 66.46 (C-4 '), 61.79 and 60.69 (C-6 and C-6 '), 54.31 (C-2), 20.69, 20.59, 20.55, 20.47, 20.47 with 20.34 (6 × OAc), 13.51 (SMe).β anomer: 1hNMR.(CDCl 3) δ: 7.48 (d, 1H, J nH, 29.68Hz, NH), 5.33 (m, 1H, H-4 '), 5.28 (dd, 1H, J 2,310.19Hz, J 3,49.12Hz, H-3), 5.02 (dd, 1H, J 1 ', 2 '7.64Hz, J 2 ', 3 '10.40Hz, H-2 '), 4.92 (dd, 1H, J 3 ', 4 '3.27Hz, H-3 '), 4.48 (m, 2H, H-1 and H-1 '), 4.15 (m, 1H, H-2), 4.04 (m, 4H, H-6 and H-6 '), 3.86 (m, 1H, H-5 '), 3.80 (m, 1H, H-4), 3.64 (m, 1H, H-5), 2.15,2.12,2.10,2.04,2.03,2.01 and 1.92 (7 × s, each 3H, 6 × OAc and SMe). 13cNMR (CDCl 3) δ: 170.97, 170.36, 170.25, 170.06, 169.94, 169.08 with 162.23 (7 × CO), (101.51 C-1 '), 92.23 (TCA), 83.03 (C-1), 76.66 with 76.66 (C-4 and C-5), 73.61 (C-3), 70.81 (C-3 '), 70.55 (C-5 '), 70.52 (C-2 '), 66.41 (C-4 ') 62.09 and 60.60 (C-6 and C-6 '), 53.62 (C-2), 20.85, 20.80, 20.57, 20.56, 20.52 with 20.40 (6 × OAc), 11.25 (SMe).
Embodiment 17
Under argon gas, by the acceptor according to embodiment 6 (compound of general formula 6, the wherein R of 10g (8.13mmol) 1=Bn, R 2=4-chlorobenzene formacyl, R 3=benzoyl, OR 1for β type) and the donor according to embodiment 16 (compound of general formula 5, the wherein R of 10g (1.6 equivalent) 4=ethanoyl ,-NR 5r 6=tribromo-acetyl base, X=SMe) be dissolved in the dry CHCl of 35ml 3in.In this solution, add 3.7gNIS and 490mgAgOTf in room temperature, and continue stir about 20min.Triethylamine (5ml) is joined in this slurry, uses CH 2cl 2(500ml) then dilution uses hypo solution (10%) to extract 2x, is separated organic phase, uses MgSO 4drying, filters, and concentrated, then this jelly carries out chromatographic separation on a silica gel column, uses CH 2cl 2: acetone gradient: 98: 2 → 95: 5.Yield: 12.7g, 80% (compound of general formula 4, wherein R 1=Bn, R 2=4-chlorobenzene formacyl, R 3=benzoyl, R 4=ethanoyl ,-NR 5r 6=-NH-tribromo-acetyl base, OR 1for in β type).MS(ESP)：1972.1[M+Na] +，1988.1[M+K] +，1948.2[M-H] -，1984.0[M+Cl] -。 13cNMR (CDCl 3) δ: 101.2,100.7,100.0,98.8 (anomeric carbon).Mp.：139-142℃。
Embodiment 18
Under argon gas, by the donor according to embodiment 11 (compound of general formula 5, the wherein R of 100g (120.4mmol) 4=ethanoyl ,-NR 5r 6=-NH-tribromo-acetyl base, X=β-SPh) and the acceptor according to embodiment 6 (compound of general formula 6, the wherein R of 118g (96.2mmol) 1=Bn, R 2=4-chlorobenzene formacyl, R 3=benzoyl, OR 1for β type) be dissolved in the dry CHCl of 250ml 3in.In room temperature, in this solution, add 38gNIS and 6gAgOTf, and continue stir about 1h.This slurry CH 2cl 2(500ml) dilution and through extracting operation.The final volume of organic phase is 1.5L, uses 450ml10%Na 2s 2o 3solution and the saturated NaHCO of 150ml 3solution.After concentration, isolate the brown jelly of 280g, it is through column chromatography, thus obtains the compound of general formula 4, wherein R 1=Bn, R 2=4-chlorobenzene formacyl, R 3=benzoyl, R 4=ethanoyl ,-NR 5r 6=-NH-tribromo-acetyl base, OR 1for β type (physical data is identical with the physical data of the compound of embodiment 17).
Embodiment 19
According to embodiment 18, prepare similarly from the donor of embodiment 11 and the acceptor of embodiment 8: the compound of general formula 4, wherein R 1=4-aminomethyl phenyl, R 2=4-chlorobenzene formacyl, R 3=benzoyl, R 4=ethanoyl ,-NR 5r 6=-NH-tribromo-acetyl base, OR 1for β type.
From the white crystal of MeOH
1h data (CDCl 3): 8.0-7.25 (m, aromatics), 6.8-7.0 (m, aromatics), 6.4 (d, 1H, NH), 5.54 (m, 2H), 5.42 (dd, 1H), 5.38 (dd, 1H), 5.3 (d, 1H), 5.04 (m, 2H), 4.92 (dd, 1H), 4.74 (d, 1H), 4.6 (m, 4H), 4.4 (m, 4H), 4.0 (m, 6H), 3.7 (m, 5H), 3.4 (m, 2H).
13c data (CDCl 3): 177.4 (NHTCA carbonyls), 170.3, 170.2, 170.1, 170.0, 169.9, 169.0 (OAc, carbonyl), 165.3, 165.1, 164.9, 164.4, 164.2, 163.5, 161.3 (OBz carbonyls), 140.2, 140.0, 139.9, 139.7, 139.4, 137.9, 133.4, 133.1, 131.2, 131.1, 130.9, 130.8, 130.7, 129.9, 129.1, 129.0, 128.9, 128.6, 128.5, 128.3, 127.9, 127.8, 127.7, 127.6, 127.5, 127.1 (aromatics carbon), 101.2, 100.7, 99.9, 98.6 (anomeric carbons), 91.7 (NHTCACCl 3), 76.2,75.4,72.9,72.8,71.7,71.6,70.8,70.7,70.5,69.5,68.9,66.5,62.8,62.1,60.9,60.7,55.7,29.5,21.1,20.6,20.5,20.4,20.3.
Embodiment 20
According to embodiment 18, prepare similarly from the donor of embodiment 11 and the acceptor of embodiment 9: the compound of general formula 4, wherein R 1=4-chloro-phenyl-, R 2=4-chlorobenzene formacyl, R 3=benzoyl, R 4=ethanoyl ,-NR 5r 6=-NH-tribromo-acetyl base, OR 1for β type.
1hNMR (CDCl 3): 7.95-6.85 (m, aromatics H), 6.42 (d, 1H), 5.55 (m), 5.4 (m, 4H), 5.0 (m, 3H), 4.75 (d, 1H), 4.55 (m, 4H), 4.4 (m, 2H), 4.0 (m), 3.7 (m, 5H), 3.45 (m, 3H).
13CNMR(CDCl 3)：170.32，170.0，169.0，165.1，164.4，163.6，161.4，140.3，139.9，139.5，134.9，133.9，133.4，131.0，130.0，128.9，128.6，127.9，127.6，127.4，127.1，101.2，100.7，99.9，99.2，91.7，76.2，75.4，73.0，71.6，70.8，70.0，69.6，68.9，68.7，66.6，65.4，63.2，62.7，62.1，60.8，55.7，53.5，30.9。
Embodiment 21
The glycosyl donor (1.0 equivalent) of embodiment 11 and the glycol acceptor (1.0 equivalent) of embodiment 2 are dissolved in 25mLCH 2cl 2in.This solution is cooled to 0 DEG C and adds NBS (1.2 equivalent).After stirring 10min, slowly add 25 μ L trifluoromethanesulfonic acids.After reaction has reached, it is operated usually.By purification by flash chromatography crude product, thus produce the compound of general formula 4, wherein R 1=benzyl, R 2=4-chlorobenzene formacyl, R 3=H, R 4=ethanoyl ,-NR 5r 6=-NH-tribromo-acetyl base, OR 1for β type (56%).
13cNMR (500MHz): 170.3, 170.1, 170.0, 169.9, 169.0, 165.0, 164.9, 164.5, 164.3, 163.6, 162.0, 140.0, 139.9, 139.8, 139.7, 139.5, 137.7, 136.3, 131.2, 131.1, 131.0, 130.8, 130.7, 129.0, 128.9 (2 signals), 128.8, 128.6, 128.5, 128.3, 128.1, 128.0 (2 signals), 127.9, 127.6 (2 signals), 127.5, 127.2, 125.2, 101.1, 100.6, 99.4, 98.7, 91.6, 79.8, 75.9, 75.4, 73.2, 73.1, 72.8, 72.1, 71.9, 71.0, 70.7, 70.6, 70.5 (2 signals), 69.0, 67.4, 66.5, 62.8, 62.7, 61.7, 60.8, 55.8, 22.6, 21.3, 20.5 (2 signals), 20.4.
Embodiment 22
10g (5.1mmol) to be dissolved in MeOH (110ml) through the tetrose (embodiment 17) of protection and to add the solution (1M, in MeOH) of NaOMe until pH10.This solution is stirred 5h at 40 DEG C, then by adding AmberliteIR120H +resin neutralizes, and by this resin filtering, filtrate is evaporated to dry.Resistates is dissolved in warm DMF (10ml) and also dropwise joins ipr 2in O (150ml), and this suspension is stirred 3h in addition.To filtering be precipitated, use ipr 2o washing (2x20ml) is also dry, thus produces product (91%) (compound of general formula 3, the wherein R of 4.2g as pale powder 1=Bn ,-NR 5r 6=-NH-tribromo-acetyl base, OR 1for β type).MS(ESP)：900.1[M-H] -。 13cNMR (D 2o) δ: 105.6,105.5,104.2,103.7 (anomeric carbons).Mp.：134，5-135℃。
Identical product can be prepared similarly from the compound of embodiment 21.
Embodiment 23
According to embodiment 22, prepare like the compounds of embodiment 19: the compound of general formula 3, wherein R 1=4-methyl-benzyl ,-NR 5r 6=-NH-tribromo-acetyl base, OR 1for β type.
1hNMR (D 2o): 7.25 (dd, 4H, aromatics), 4.98 (d, 1H), 4.88 (d, 1H), 4.8 (s), 4.7 (d, 1H), 4.5 (dd, 1H), 4.44 (d, 1H), 4.18d (1H), 3.9 (m), 3.78 (m), 3.6 (m), 3.36 (s).
13CNMR(D 2O)：167.7，141.5，136.2，132.0，131.7，105.7，105.6，104.2，103.7，94.3，84.2，81.1，80.9，78.1，77.6，77.5，77.4，77.1，75.5，75.3，74.5，74.1，73.7，72.9，71.3，71.0，63.8，63.7，62.9，62.6，60.0，51.6，23.0。
Embodiment 24
According to embodiment 22, prepare like the compounds of embodiment 20: the compound of general formula 3, wherein R 1=4-chlorobenzyl ,-NR 5r 6=-NH-tribromo-acetyl base, OR 1for β type.
1hNMR (D 2o): 7.2 (m, 4H, aromatics), 4.96 (d, 1H), 4.88 (d, 1H), 4.72 (d, 1H), 4.62 (d, 1H), 4.52 (m, 2H), 4.42 (m, 1H), 4.16 (d, 1H), 3.9 (m), 3.7 (m), 3.65 (m), 3.58 (m).
13cNMR (D 2o): 167.7 (NHTCA carbonyls), 137.9,136.2,132.9,131.3,105.7,105.6,104.3,103.8 (4x anomeric carbon), 94.3 (NHTCACCl 3), 84.2,81.4,81.1,80.9,78.1,77.6,77.5,77.4,77.1,75.5,75.3,74.6,74.5,73.7,73.4,72.9,71.3,71.0,63.8,63.7,62.8,62.6,60.2,60.0,59.8.
Embodiment 25
By the compound dissolution of the embodiment 22 of 35g in 110mlMeOH and 110mlKOH (7.5g) aqueous solution and by this mixture stirring at room temperature 1 day.Then profit is cooled with an ice bath this mixture, is neutralized and be concentrated into dry by HCl gas.Then at room temperature pyridine (150ml) and diacetyl oxide (150ml) by thick amber glass shape thing (compound of general formula 2, wherein R 1=Bn, OR 1for β type, 13cNMR (D 2o) δ: 107.1,105.7,105.4,103.7 (anomeric carbons)) acetylize 1 day.Solution is concentrated, this slurry is dissolved in CH 2cl 2in, by 1MHCl solution extraction organic phase, then use saturated NaHCO 3solution extraction organic phase, uses MgSO 4drying, filters and concentrates, thus produces 43g brown colored foams.Make it through column chromatography, thus obtain the compound of general formula 4a, wherein R 1=Bn, OR 1for β type). 13cNMR (CDCl 3) δ: 101.2,100.8,100.4,99.2 (anomeric carbon).
Embodiment 26
Go-N-acylations step according to embodiment 25, prepare like the compounds of embodiment 23: the compound of general formula 2, wherein R 1=4-methyl-benzyl, OR 1for β type.
1hNMR (D 2o): 7.3 (dd, 4H, aromatics), 4.9 (d, 1H), 4.72 (dd, 1H), 4.5 (m), 4.18 (d, 1H), 3.95 (m), 3.8-3.55 (m), 3.34 (dd).
13CNMR(D 2O)：184.1，141.4，136.6，136.1，131.9，131.6，131.4，105.7，105.3，105.2，100.0，84.8，81.0，80.9，78.0，77.7，77.5，77.4，77.1，75.9，75.5，75.2，74.4，74.0，73.6，73.2，72.7，72.4，71.2，70.9，63.7，63.6，62.5，58.9，25.9。
Embodiment 27
Go-N-acylations step according to embodiment 25, prepare like the compounds of embodiment 24: the compound of general formula 2, wherein R 1=4-chlorobenzyl, OR 1for β type.
1hNMR (D 2o): 7.4 (s, 4H, aromatics), 4.9 (m, 2H), 4.75 (m), 4.52 (dd, 1H), 4.46 (dd, 1H), 4.18 (d, 1H), 3.96 (m), 3.86 (m), 3.7 (m), 3.62 (m).
13CNMR(D 2O)：184.1，137.9，136.2，132.9，132.6，131.3，105.7，105.2，104.0，103.8，84.6，80.9，80.7，78.1，77.6，77.4，77.1，75.5，75.2，74.4，73.6，73.4，72.7，71.2，70.9，63.8，63.6，62.8，62.4，58.6，51.6，25.9。
Embodiment 28
140g (107.5mmol) is crossed acetylizad tetrose (embodiment 25) to be dissolved in 1.5LMeOH, adds NaOMe solution (1M) until pH10, and is spent the night 50 DEG C of stirrings by this mixture.Product crystallization from this reaction mixture.Allow this mixture to be cooled to room temperature, then cooled, filter, with cold EtOH wash filtrate, then dry, thus produce 69g white powder (compound of general formula 1, wherein R 1=Bn, OR 1for β type; 80%). 13cNMR (D 2o) δ: 105.6,105.5,105.4,103.6 (anomeric carbons).Mp.：284-286℃。
Embodiment 29
In room temperature, by amine (compound of general formula 2, the R of 150g purifying 1=benzyl, OR 1for β type) join in the mixture of water (150mL) and MeOH (200mL).Add the diacetyl oxide (20mL) of a part.After the stirring of 1h, add extra 10mL diacetyl oxide and by mixture in stirring at room temperature.After the stirring of 1h, add extra 10mL diacetyl oxide and mixture is stirred 2h again in room temperature.Finally, mixture is crystalline material.Add MeOH (250mL) and mixture is put into refrigerator overnight.Filtering solids, with cold MeOH (200mL) washing leaching cake, thus produces 260g brown solid.This solid at 50 DEG C under atmospheric pressure dry (3 days), produces 118g product (compound of general formula 1, R 1=Bn, OR 1for β type).The recrystallize of 80g in MeOH/ water produces the 72.6g product with 99.7% purity (according to HPLC).
Embodiment 30
According to embodiment 29, prepare like the compounds of embodiment 26: the compound of general formula 1, R 1=4-methyl-benzyl, OR 1for β type.
1HNMR(D 2O)：7.3(dd，4H)，4.88(d，1H)，4.7(m)，4.54(d，1H)，4.48(d，1H)，4.42(d，1H)，4.34(d)，4.0-3.5(m)，3.34(dd，1H)。
13CNMR(D 2O)：184.2，177.6，173.7，141.5，136.1，131.9，131.4，105.6，105.5，105.4，103.6，93.2，84.7，81.5，81.0，80.8，78.0，77.6，77.4，77.1，75.5，75.2，74.8，74.0，73.6，72.9，63.7，62.8，58.9，56.4，25.9，22.9。
Embodiment 31
According to embodiment 29, prepare like the compounds of embodiment 27: the compound of general formula 1, R 1=4-chlorobenzyl, OR 1for β type.
1HNMR(D 2O)：7.4(s，4H)，4.9(d，1H)，4.72(m)，4.52(d，1H)，4.8(d，1H)，4.42(d，1H)，4.16(d，1H)，4.0-3.52(m)。
13CNMR(D 2O)：138.9，177.6，138.3，137.9，136.2，131.3，105.6，105.5，105.4，103.7，93.2，86.1，84.7，81.5，81.0，80.8，78.0，77.5，77.4，77.2，77.1，75.5，75.2，74.9，63.7，58.9，57.8，56.4，24.8。
Embodiment 32
40g (50.1mmol) benzyl glucosides (embodiment 28 or 29) to be dissolved in 200ml water, 1.6gPd-C and to add 400 μ l acetic acid, and by this mixture in room temperature at H 2stir 2 days under atmosphere (about 40bar).Filtration catalizer, washes filter cake with water, and filtrate is dropwise joined in 1.6l acetone, then cools, and filters and by dry under vacuo for the solid collected, thus produces the white powder (44.5mmol, 89%) of 31.5gLNnT.
Embodiment 33
Benzyl glucosides (embodiment 28 or 29,100g) to be dissolved in 300ml water, 2ml acetic acid and to add 30mlMeOH, then adding 10gPd-C (5%).In room temperature, by the hydrogenation 2 days under the hydrogen of 5bar of this mixture.Gac (5g) is joined in this mixture, filtration catalizer and carbon, dilute filtrate with water (200ml), this aqueous solution is heated to 50 DEG C.Crystallization is carried out as described above by adding EtOH (4L).Be separated the white solid obtaining 65gLNnT.
Embodiment 34
50g benzyl glucosides (embodiment 28 or 29) is dissolved in 110mL water and also dilutes this solution with MeOH (120mL).Add Pd-C (2g, 10%Pd) catalyzer, and at 60 DEG C at H 2this mixture of (5bar) hydrogenation under atmosphere.After 7h, filtration catalizer, uses about 20mL water: MeOH mixture (1: 1) washing leaching cake.
Crystallization: by warm for the solution obtained above (50-55 DEG C), be cooled to gradually in the process of 40 DEG C divide wherein 3-5 part add heat MeOH (600mL).Crystallization starts immediately.After adding methyl alcohol, crystalline mixture slow (3h) is cooled to room temperature, then puts into refrigerator overnight.Glass filter filters, washs solid with cold MeOH, and 60 DEG C in vacuum drying oven dry 2 days, thus produce 37.4g white solid.HPLC purity: 100%, mp.:226-230 DEG C. 13c-NMR (600MHz, D 2o: CD 3oD=3: 2) δ: Glc (α) C-193.2C-272.5C-372.7C-479.5C-571.3C-661.2, Glc (β) C-197.2C-275.2C-375.7C-479.5C-576.1C-661.3, GalC-1104.3C-271.4C-383.1C-469.7C-5*76.2C-662.3, GlcNAcC-1104.1C-256.4C-373.4C-479.2C-575.8C-661.0NCOCH 323.3174.8, GalC-1104.2C-272.3C-373.9C-469.9C-5*76.6C-662.3 (the interchangeable distribution of *).

Claims (14)

1. the polymorphic form of a Galp β 1-4GlcNAcp β 1-3Galp β 1-4Glc, it is characterized in that, based on the alpha-emitting measurement of use CuK, described polymorphic form is presented at 20.32 ± 0.20,19.10 ± 0.20,7.98 ± 0.20, the X-ray powder diffraction reflection at 21.03 ± 0.20,20.95 ± 0.20 and 5.66 ± 0.202 Θ angles.
2. the polymorphic form of Galp β 1-4GlcNAcp β 1-3Galp β 1-4Glc according to claim 1, described polymorphic form is presented at the fusing point between 226-230 DEG C.
3. the polymorphic form of the Galp β 1-4GlcNAcp β 1-3Galp β 1-4Glc according to any one of claim 1 and 2, it is used as medicine.
4. the polymorphic form of the Galp β 1-4GlcNAcp β 1-3Galp β 1-4Glc according to any one of claim 1 and 2, it is used as nutritional additive.
5. a pharmaceutical composition, described pharmaceutical composition comprises the polymorphic form of the Galp β 1-4GlcNAcp β 1-3Galp β 1-4Glc of definition any one of claim 1 and 2.
6. a nutritive compositions, described nutritive compositions comprises the polymorphic form of the Galp β 1-4GlcNAcp β 1-3Galp β 1-4Glc of definition any one of claim 1 and 2.
7. one kind for from comprising one or more C 1-C 6the method of the polymorphic form of crystallization Galp β 1-4GlcNAcp β 1-3Galp β 1-4Glc in the solvent systems of alcohol, based on the alpha-emitting measurement of use CuK, described polymorphic form is presented at 20.32 ± 0.20,19.10 ± 0.20,7.98 ± 0.20, the X-ray powder diffraction reflection at 21.03 ± 0.20,20.95 ± 0.20 and 5.66 ± 0.202 Θ angles.
8. method according to claim 7, wherein said solvent systems comprises water.
9. method according to claim 8, wherein said solvent systems is water/methyl alcohol.
10. method according to claim 9, it comprises the steps:
A) use water/Methanol for Galp β 1-4GlcNAcp β 1-3Galp β 1-4Glc solution,
B) described Galp β 1-4GlcNAcp β 1-3Galp β 1-4Glc solution is warming to 50-60 DEG C,
C) be cooled at 35-45 DEG C gradually, in described Galp β 1-4GlcNAcp β 1-3Galp β 1-4Glc solution, adding hot methanol,
D) kind of a crystalline substance is optionally added,
E) the warm suspension of gained is cooled to 0-8C.
11. method according to claim 10, wherein step a) in water/carbinol mixture used be ≈ 1: 1 mixture.
12. method according to claim 10, wherein step a) in Galp β 1-4GlcNAcp β 1-3Galp β 1-4Glc solution there is the concentration of 140-180g/L.
13. method according to claim 10, wherein step c) in, the 115-250% of the initial volume of hot methanol as many as added.
14. methods according to claim 10, wherein step a) in water/carbinol mixture be ≈ 1: 1 mixture, step a) in Galp β 1-4GlcNAcp β 1-3Galp β 1-4Glc solution there is the concentration of 140-180g/L, and in step c) in, the 115-250% of the initial volume of hot methanol as many as added.
CN201510751641.5A 2010-02-19 2011-02-21 Method for preparation of tetrasaccharide lacto-N-neotetraose (LNNT) containing N-acetyllactosamine Pending CN105330705A (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DKPA201070060 2010-02-19
DKPA201070060 2010-02-19
CN2011800097783A CN102803282A (en) 2010-02-19 2011-02-21 A method for preparation of the tetrasaccharide lacto-N-neotetraose (lNNT) containing N-acetyllactosamine

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
CN2011800097783A Division CN102803282A (en) 2010-02-19 2011-02-21 A method for preparation of the tetrasaccharide lacto-N-neotetraose (lNNT) containing N-acetyllactosamine

Publications (1)

Publication Number Publication Date
CN105330705A true CN105330705A (en) 2016-02-17

Family

ID=44482456

Family Applications (2)

Application Number Title Priority Date Filing Date
CN2011800097783A Pending CN102803282A (en) 2010-02-19 2011-02-21 A method for preparation of the tetrasaccharide lacto-N-neotetraose (lNNT) containing N-acetyllactosamine
CN201510751641.5A Pending CN105330705A (en) 2010-02-19 2011-02-21 Method for preparation of tetrasaccharide lacto-N-neotetraose (LNNT) containing N-acetyllactosamine

Family Applications Before (1)

Application Number Title Priority Date Filing Date
CN2011800097783A Pending CN102803282A (en) 2010-02-19 2011-02-21 A method for preparation of the tetrasaccharide lacto-N-neotetraose (lNNT) containing N-acetyllactosamine

Country Status (6)

Country Link
US (2) US8993740B2 (en)
EP (2) EP3241836A1 (en)
CN (2) CN102803282A (en)
DE (1) DE202011111124U1 (en)
HK (1) HK1246302A1 (en)
WO (1) WO2011100980A1 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109891173A (en) * 2016-10-31 2019-06-14 格礼卡姆股份公司 The method for removing residual organic solvents from crystallization oligosaccharides
CN111925403A (en) * 2019-05-13 2020-11-13 山东大学 Glucosamine donors and application thereof

Families Citing this family (57)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9102966B2 (en) 2010-07-16 2015-08-11 Glycom A/S Synthesis of sialooligosaccharide derivatives
RU2013155453A (en) 2011-05-13 2015-06-20 Глюком А/С DIVERSIFICATION OF HUMAN MILK OLIGOSACCHARIDES (HMOS) OR THEIR PRECEDENTS
EP2707493A4 (en) * 2011-05-13 2014-10-08 Glycom As METHOD FOR GENERATING HUMAN MILK OLIGOSACCHARIDES (HMOs) OR PRECURSORS THEREOF
WO2013044928A1 (en) * 2011-09-30 2013-04-04 Glycom A/S Synthesis of hmo core structures
US20140271562A1 (en) * 2011-10-18 2014-09-18 Nestec S.A. Composition for use in brain growth and/or cognitive and/or psychomotor development
EP2859112A4 (en) 2012-06-08 2015-10-28 Glycom As Method for producing oligosaccharides and oligosaccharide glycosides by fermentation
CN104428307A (en) * 2012-06-14 2015-03-18 格礼卡姆股份公司 Enhancing the stability and purity and increasing the bioavailability of human milk oligosaccharides or precursors or blends thereof
US20150182549A1 (en) * 2012-06-22 2015-07-02 Glycom A/S Method for enzymatic glycosylation of oligosaccharides from mammalian animal milk
CN102942598B (en) * 2012-11-28 2015-07-01 济南圣泉唐和唐生物科技有限公司 Post-treatment method for acetyl sugar halogenating reaction
WO2014086373A1 (en) * 2012-12-07 2014-06-12 Glycom A/S Crystallisation of human milk oligosaccharides (hmo)
EP2935300A4 (en) * 2012-12-18 2016-06-01 Glycom As Novel polymorphs of lnnt
US20160113952A1 (en) 2013-05-22 2016-04-28 Glycom As Synthetic Mixture of Oligosaccharides for Treating a Microbiota of a Mammal
CN104231009B (en) * 2013-06-08 2017-07-04 上海医药工业研究院 A kind of levulan, its intermediate, Preparation method and use
US10364449B2 (en) 2013-09-06 2019-07-30 Glycom A/S Fermentative production of oligosaccharides
EP3209673B2 (en) 2014-10-24 2023-07-26 Glycom A/S Mixtures of human milk oligosaccharides
PL3212198T3 (en) 2014-10-29 2021-08-23 Glycom A/S Synthetic composition and method for promoting mucosal healing
US11040049B2 (en) 2014-10-29 2021-06-22 Glycom A/S Composition comprising HMSs/HMOs and use thereof
WO2016066175A1 (en) 2014-10-29 2016-05-06 Glycom A/S Synthetic composition and method for treating irritable bowel syndrome
US11040050B2 (en) 2014-10-29 2021-06-22 Glycom A/S Composition comprising HMSs/HMOs and use thereof
WO2016086947A1 (en) * 2014-12-05 2016-06-09 Glycom A/S Crystalline difucosyllactose
US10835544B2 (en) 2014-12-08 2020-11-17 Glycom A/S Synthetic composition for regulating satiety
US10881674B2 (en) 2014-12-08 2021-01-05 Glycom A/S Synthetic composition for treating metabolic disorders
US10828313B2 (en) 2014-12-08 2020-11-10 Glycom A/S Synthetic composition for treating metabolic disorders
US10987368B2 (en) 2014-12-08 2021-04-27 Glycom A/S Synthetic composition for preventing or treating CVD
DE202016008919U1 (en) 2015-03-05 2020-09-07 Société des Produits Nestlé S.A. Compositions for use in the prevention or treatment of gastrointestinal infection / inflammation in infants or young children
ES2960750T3 (en) 2015-03-05 2024-03-06 Glycom As Human milk oligosaccharides for the treatment of acute respiratory tract infections
WO2016157108A1 (en) 2015-03-31 2016-10-06 Glycom A/S Mixtures of human milk oligosaccharides comprising 3 '-o-sialyllactose
CN107614511B (en) 2015-06-09 2020-10-23 格礼卡姆股份公司 Ternary mixtures of 6' -SL, LNnT and LST c
DK3349763T3 (en) 2015-09-14 2021-11-15 Glycom As Composition for use in microbiota modulation
EP3368046B1 (en) 2015-10-28 2022-07-27 Glycom A/S Synthetic composition and method for modulating brain function and behaviour
ES2950489T3 (en) 2015-10-28 2023-10-10 Glycom As Synthetic composition and method to modulate emotion and mood disorders
CN108348536B (en) 2015-11-17 2021-09-24 格礼卡姆股份公司 Synthetic compositions for treating antibiotic-associated complications
US11957148B2 (en) 2015-12-15 2024-04-16 Societe Des Produits Nestle S.A. Mixture of human milk oligosaccharides(HMOs)
EP3419630A4 (en) 2016-02-24 2019-11-06 Glycom A/S Synthetic composition for microbiota modulation
ES2938355T3 (en) 2016-05-05 2023-04-10 Glycom As Composition comprising HMOS for the treatment of non-infectious diarrhea
ES2924137T3 (en) 2016-05-05 2022-10-04 Glycom As Composition comprising HMOS for use in the treatment of hypersensitivity and/or visceral pain mediated by mast cells
EP3458073B1 (en) 2016-05-19 2023-10-25 Glycom A/S Use of human milk oligosaccharides for treating migraine in patients with inflammatory bowel syndrome
WO2017215722A1 (en) * 2016-06-15 2017-12-21 Glycom A/S Synthetic composition comprising hmo and method for modulating emotion and mood disorders
EP3474861A4 (en) 2016-06-24 2020-04-15 Glycom A/S Compositions comprising hmos, their production and use for the prevention and/or treatment of viral and/or bacterial infections
EP3589139A4 (en) 2017-03-01 2020-12-23 Glycom A/S Synthetic composition for microbiota modulation
EP3634429A4 (en) 2017-05-09 2020-11-04 Glycom A/S Synthetic composition for microbiota modulation
US11541068B2 (en) 2017-05-24 2023-01-03 Glycom A/S HMO compositions and methods for reducing autism spectrum disorder symptoms
WO2018215960A1 (en) 2017-05-24 2018-11-29 Glycom A/S Synthetic composition comprising one or more human milk oligosaccharides (hmos)
WO2019012461A1 (en) 2017-07-12 2019-01-17 Glycom A/S Amorphous mixture comprising a neutral mono- or oligosaccharide and an acidic non-carbohydrate component
EP3672600A4 (en) 2017-08-21 2021-05-19 Glycom A/S Synthetic composition for reducing allergy symptoms
WO2019087140A1 (en) 2017-11-02 2019-05-09 Glycom A/S One or more hmos for reducing or preventing fatigue and/or improving focus or concentration
JP7327724B2 (en) 2017-11-30 2023-08-16 グリコム・アクティーゼルスカブ A mixture of HMOs to treat wheat sensitivity
US11602545B2 (en) 2017-12-05 2023-03-14 Glycom A/S Human milk oligosaccharides for treating migraine
EP3743078A4 (en) 2017-12-22 2022-01-19 Glycom A/S COMPOSITION COMPRISING HMOs FOR PREVENTING OR REDUCING NOCICEPTION
CN112165947A (en) 2018-05-31 2021-01-01 格礼卡姆股份公司 HMO mixtures for the treatment of autoimmune diseases
KR20210104773A (en) 2018-12-19 2021-08-25 글리콤 에이/에스 Compositions and methods for the treatment of humans using a low-FODMAP diet
EP3750902A1 (en) * 2019-06-14 2020-12-16 Jennewein Biotechnologie GmbH Process for the purification of lacto-n-neotetraose
CN116261398A (en) 2020-10-16 2023-06-13 雀巢产品有限公司 Nutritional composition comprising human milk oligosaccharides
EP4243634A1 (en) 2020-11-10 2023-09-20 Société des Produits Nestlé S.A. Nutritional composition
WO2022223430A1 (en) 2021-04-19 2022-10-27 Dsm Ip Assets B.V. A composition of enzymes and human milk oligosaccharides
DK202101233A1 (en) 2021-12-21 2023-06-27 Dsm Ip Assets Bv Crystallization of LNnT
WO2023141513A2 (en) * 2022-01-19 2023-07-27 The Regents Of The University Of California Functionalized human milk oligosaccharides and methods for producing them

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1503803A (en) * 2001-06-11 2004-06-09 协和发酵工业株式会社 Crystals of oligosaccharides and processes for preparation thereof
CN1671867A (en) * 2002-06-27 2005-09-21 达尼斯科甜味剂股份有限公司 Crystallization of sugars
US20090098240A1 (en) * 2007-05-17 2009-04-16 The Regents Of The University Of California Human milk oligosaccharides to promote growth of beneficial gut bacteria

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0870841A4 (en) 1996-09-17 2001-04-11 Kyowa Hakko Kogyo Kk Processes for producing sugar nucleotides and complex carbohydrates
JP3100559B2 (en) 1997-02-26 2000-10-16 ヤマサ醤油株式会社 Process for producing lacto-N-neotetraose
CN1141309C (en) * 1999-11-25 2004-03-10 中国科学院生态环境研究中心 Preparation method of pyranoligosaccharide with 1-6 connection and 1,2-trans glycosidic band
CN1129600C (en) * 1999-12-16 2003-12-03 中国科学院生态环境研究中心 Synthesis of lentinan core fragment trisaccharide, tetrasaccharide, hexasaccharide and heptasaccharide
US6960654B2 (en) * 2002-06-11 2005-11-01 The Board Of Trustees Of The University Of Illinois Method of forming glycosidic bonds from thioglycosides using an N,N-dialkylsulfinamide
CN101168550A (en) * 2006-10-25 2008-04-30 中国科学院大连化学物理研究所 Method for synthesizing aminoglucose tetrasaccharide

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1503803A (en) * 2001-06-11 2004-06-09 协和发酵工业株式会社 Crystals of oligosaccharides and processes for preparation thereof
CN1671867A (en) * 2002-06-27 2005-09-21 达尼斯科甜味剂股份有限公司 Crystallization of sugars
US20090098240A1 (en) * 2007-05-17 2009-04-16 The Regents Of The University Of California Human milk oligosaccharides to promote growth of beneficial gut bacteria

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ANGELIKA ANDERSON，等: "Improved method for the isolation of 2’-fucosyllactose from human milk", 《JOURNAL OF CHROMATOGRAPH》 *
KUHN，等: "Die Konstitution der Lacto-N-neotetraose", 《EUROPEAN JOURNAL OF INORGANIC CHEMISTRY》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109891173A (en) * 2016-10-31 2019-06-14 格礼卡姆股份公司 The method for removing residual organic solvents from crystallization oligosaccharides
CN111925403A (en) * 2019-05-13 2020-11-13 山东大学 Glucosamine donors and application thereof

Also Published As

Publication number Publication date
CN102803282A (en) 2012-11-28
DE202011111124U1 (en) 2020-05-22
WO2011100980A1 (en) 2011-08-25
US8993740B2 (en) 2015-03-31
EP2536737A1 (en) 2012-12-26
EP2536737A4 (en) 2013-10-02
US20120309949A1 (en) 2012-12-06
EP2536737B1 (en) 2017-05-31
US20150239923A1 (en) 2015-08-27
HK1246302A1 (en) 2018-09-07
EP3241836A1 (en) 2017-11-08

Similar Documents

Publication Publication Date Title
CN105330705A (en) Method for preparation of tetrasaccharide lacto-N-neotetraose (LNNT) containing N-acetyllactosamine
US9237764B2 (en) Production of 6′-O-sialyllactose and intermediates
EP2417143B1 (en) Synthesis of 2&#39;-o-fucosyllactose
EP2678347A1 (en) Catalytic hydrogenolysis of a composition of a mixture of oligosaccharide precursors and uses thereof
WO2013091660A1 (en) A method for obtaining crystalline lacto-n-tetraose and lacto-n-neotetraose precursors and mixtures thereof
CN103703012A (en) Manufacture of lacto-n-tetraose
EP2828275A1 (en) Synthesis of the trisaccharide 3-o-fucosyllactose and intermediates thereof
CN103958537A (en) Synthesis of HMO core structures
US10500221B2 (en) Crystalline difucosyllactose
CN103443113A (en) Synthesis of new fucose-containing carbohydrate derivatives
CN103025749A (en) Polymorphs of 2&#39;-o-fucosyllactose and producing thereof
CN104812768A (en) Crystalline 3-O-fucosyllactose
KR20120004504A (en) 6&#34;-SIALYLLACTOSE SALTS AND PROCESS FOR THEIR SYNTHESIS AND FOR THE SYNTHESIS OF OTHER &amp;alpha;-SIALYLOLIGOSACCHARIDES
Verma et al. International Journal of Carbohydrate Research
CN110818750A (en) Synthesis method of stevioside R

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication

Application publication date: 20160217

RJ01 Rejection of invention patent application after publication